Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to secreted and transmembrane polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

[0002] Extracellular proteins play important roles in, among otherthings, the formation, differentiation and maintenance of multicellularorganisms. The fate of many individual cells, e.g., proliferation,migration, differentiation, or interaction with other cells, istypically governed by information received from other cells and/or theimmediate environment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

[0003] Secreted proteins have various industrial applications, includingas pharmaceuticals, diagnostics, biosensors and bioreactors. Mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane proteins, also have potential as therapeutic ordiagnostic agents. Efforts are being undertaken by both industry andacademia to identify new, native secreted proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);U.S. Pat. No. 5,536,637)].

[0004] Membrane-bound proteins and receptors can play important rolesin, among other things, the formation, differentiation and maintenanceof multicellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

[0005] Membrane-bound proteins and receptor molecules have variousindustrial applications, including as pharmaceutical and diagnosticagents. Receptor immunoadhesins, for instance, can be employed astherapeutic agents to block receptor-ligand interactions. Themembrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction.

[0006] Efforts are being undertaken by both industry and academia toidentify new, native receptor or membrane-bound proteins. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

[0007] 1. PRO241

[0008] Cartilage is a specialized connective tissue with a largeextracellular matrix containing a dense network of collagen fibers and ahigh content of proteoglycan. While the majority of the proteoglycan incartilage is aggrecan, which contains many chondroitin sulphate andkeratin sulphate chains and forms multimolecular aggregates by bindingwith link protein to hyaluronan, cartilage also contains a number ofsmaller molecular weight proteoglycans. One of these smaller molecularweight proteoglycans is a protein called biglycan, a proteoglycan whichis widely distributed in the extracellular matrix of various otherconnective tissues including tendon, sclera, skin, and the like.Biglycan is known to possess leucine-rich repeat sequences and twochondroitin sulphate/dermatan sulphate chains and functions to bind tothe cell-binding domain of fibronectin so as to inhibit cellularattachment thereto. It is speculated that the small molecular weightproteoglycans such as biglycan may play important roles in the growthand/or repair of cartilage and in degenrative diseases such asarthritis. As such, there is an interest in identifying andcharacterizing novel polypeptides having homology to biglycan protein.

[0009] We herein describe the identification and characterization ofnovel polypeptides having homology to the biglycan protein, whereinthose polypeptides are herein designated PRO241 polypeptides.

[0010] 2. PRO243

[0011] Chordin (Xenopus, Xchd) is a soluble factor secreted by theSpemann organizer which has potent dorsalizing activity (Sasai et al.,Cell 79: 779-90 (1994); Sasai et al., Nature 376: 333-36 (1995). Otherdorsalizing factors secreted by the organizer are noggin (Smith andHarlan, Cell 70: 829-840 (1992); Lamb et al, Science 262: 713-718 (1993)and follistatin (Hemmanti-Brivanlou et al., Cell 77: 283-295 (1994).Chordin subdivides primitive ectoderm into neural versus non-neuraldomains, and induces notochord and muscle formation by the dorsalizationof the mesoderm. It does this by functioning as an antagonist of theventralizing BMP4 signals. This inhibition is mediated by direct bindingof chordin to BMP-4 in the extracellular space, thereby preventing BMP-4receptor activation by BMP4 (Piccolo et al., Develop. Biol. 182: 5-20(1996).

[0012] BMP4 is expressed in a gradient from the ventral side of theembryo, while chordin is expressed in a gradient complementary to thatof BMP-4. Chordin antagonizes BMP-4 to establish the low end of theBMP-4 gradient. Thus, the balance between the signal from chordin andother organizer-derived factors versus the BMP signal provides theectodermal germ layer with its dorsal-ventral positional information.Chordin may also be involved in the dorsal-ventral patterning of thecentral nervous system (Sasai et al, Cell 79: 779-90 (1994). It alsoinduces exclusively anterior neural tissues (forebrain-type), therebyanteriorizing the neural type (Sasai et al, Cell 79: 779-90 (1997).Given its role in neuronal induction and patterning, chordin may proveuseful in the treatment of neurodegenerative disorders and neuraldamage, e.g., due to trauma or after chemotherapy.

[0013] We herein describe the identification and characterization ofnovel polypeptides having homology to the chordin protein, wherein thosepolypeptides are herein designated PRO243 polypeptides.

[0014] 3. PRO299

[0015] The notch proteins are involved in signaling during development.They may effect asymmetric development potential and may signalexpression of other proteins involved in development. [See Robey, E.,Curr. Opin. Genet. Dev., 7(4):551 (1997), Simpson, P., Curr. Opin.Genet. Dev., 7(4):537 (1997) Blobel, C P., Cell, 90(4):589 (1997)],Nakayama, H. et al., Dev. Genet., 21(1):21 (1997), Nakayama, H. et al.,Dev. Genet., 21(1):21 (1997), Sullivan, S. A. et al., Dev. Genet.,20(3):208 (1997) and Hayashi, H. et. al., Int. J. Dev. Biol.,40(6):1089(1996).] Serrate-mediated activation of notch has beenobserved in the dorsal compartment of the Drosophila wing imaginal disc.Fleming et al., Development, 124(15):2973 (1997). Notch is of interestfor both its role in development as well as its signaling abilities.Also of interest are novel polypeptides which may have a role indevelopment and/or signaling.

[0016] We herein describe the identification and characterization ofnovel polypeptides having homology to the notch protein, wherein thosepolypeptides are herein designated PRO299 polypeptides.

[0017] 4. PRO323

[0018] Dipeptidases are enzymatic proteins which function to cleave alarge variety of different dipeptides and which are involved in anenormous number of very important biological processes in mammalian andnon-mammalian organisms. Numerous different dipeptidase enzymes from avariety of different mammalian and non-mammalian organisms have beenboth identified and characterized. The mammalian dipeptidase enzymesplay important roles in many different biological processes including,for example, protein digestion, activation, inactivation, or modulationof dipeptide hormone activity, and alteration of the physical propertiesof proteins and enzymes.

[0019] In light of the important physiological roles played bydipeptidase enzymes, efforts are being undertaken by both industry andacademia to identify new, native dipeptidase homologs. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted and membrane-boundreceptor proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93-7108-7113 (1996); U.S. Pat. No. 5,536,637)].

[0020] We herein describe the identification and characterization ofnovel polypeptides having homology to various dipeptidase enzymes,designated herein as PRO323 polypeptides.

[0021] -b 5. PRO327

[0022] The anterior pituitary hormone prolactin is encoded by a memberof the growth hormone/prolactin/placental lactogen gene family. Inmammals, prolactin is primarily responsible for the development of themammary gland and lactation. Prolactin functions to stimulate theexpression of milk protein genes by increasing both gene transcriptionand mRNA half-life.

[0023] The physiological effects of the prolactin protein are mediatedthrough the ability of prolactin to bind to a cell surface prolactinreceptor. The prolactin receptor is found in a variety of different celltypes, has a molecular mass of approximately 40,000 and is apparentlynot linked by disulfide bonds to itself or to other subunits. Prolactinreceptor levels are differentially regulated depending upon the tissuestudied.

[0024] Given the important physiological roles played by cell surfacereceptor molecules in vivo, efforts are currently being undertaken byboth industry and academia to identify new, native membrane-boundreceptor proteins, including those which share sequence homology withthe prolactin receptor. Many of these efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel membrane-bound receptor proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);U.S. Pat. No. 5,536,637)].

[0025] We herein describe the identification and characterization ofnovel polypeptides having significant homology to the prolactin receptorprotein, designated herein as PRO327 polypeptides.

[0026] 6. PRO233

[0027] Studies have reported that the redox state of the cell is animportant determinant of the fate of the cell. Furthermore, reactiveoxygen species have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons, including the control and prevention of strokes, heartattacks, oxidative stress and hypertension.

[0028] Oxygen free radicals and antioxidants appear to play an importantrole in the central nervous system after cerebral ischemia andreperfusion. Moreover, cardiac injury, related to ischaemia andreperfusion has been reported to be caused by the action of freeradicals. In this regard, reductases, and particularly, oxidoreductases,are of interest. In addition, the transcription factors, NF-kappa B andAP-1, are known to be regulated by redox state and to affect theexpression of a large variety of genes thought to be involved in thepathogenesis of AIDS, cancer, atherosclerosis and diabeticcomplications. Publications further describing this subject matterinclude Kelsey et al., Br. J. Cancer, 76(7):852-854 (1997); Friedrichand Weiss, J. Theor. Biol., 187(4):529-540 (1997) and Pieulle et al., J.Bacteriol., 179(18):5684-5692 (1997). Given the physiological importanceof redox reactions in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in redox reactions. Wedescribe herein the identification and characterization of novelpolypeptides which have homology to reductase, designated herein asPRO233 polypeptides.

[0029] 7. PRO344

[0030] The complement proteins comprise a large group of serum proteinssome of which act in an enzymatic cascade, producing effector moleculesinvolved in inflammation. The complement proteins are of particularphysiological importance in regulating movement and function of cellsinvolved in inflammation. Given the physiological importance ofinflammation and related mechanisms in vivo, efforts are currently beingunder taken to identify new, native proteins which are involved ininflamation. We describe herein the identification and characterizationof novel polypeptides which have homology to complement proteins,wherein those polypeptides are herein designated as PRO344 polypeptides.

[0031] 8. PRO347

[0032] Cysteine-rich proteins are generally proteins which haveintricate three-dimensional structures and/or exist in multimeric formsdue to the presence of numerous cysteine residues which are capable offorming disulfide bridges. One well known cysteine-rich protein is themannose receptor which is expressed in, among other tissues, liver whereit serves to bind to mannose and transport it into liver cells. Othercysteine-rich proteins are known to play important roles in many otherphysiological and biochemical processes. As such, there is an interestin identifying novel cysteine-rich proteins. In this regard, Applicantsdescribe herein the identification and characterization of novelcysteine-rich polypeptides that has significant sequence homology to thecysteine-rich secretory protein-3, designated herein as PRO347polypeptides.

[0033] 9. PRO354

[0034] Inter-alpha-trypsin inhibitor (ITI) is a large (Mr approximately240,000) circulating protease inhibitor found in the plasma of manymammalian species. The intact inhibitor is a glycoprotein and consistsof three glycosylated subunits that interact through a strongglycosaminoglycan linkage. The anti-trypsin activity of ITI is locatedon the smallest subunit (i.e., the light chain) of the complex, whereinthat light chain is now known as the protein bikunin. The mature lightchain consists of a 21-amino acid N-terminal sequence, glycosylated atSer-10, followed by two tandem Kunitz-type domains, the first of whichis glycosylated at Asn45 and the second of which is capable ofinhibiting trypsin, chymotrypsin and plasmin. The remaining two chainsof the ITI complex are heavy chains which function to interact with theenzymatically active light chain of the complex.

[0035] Efforts are being undertaken by both industry and academia toidentify new, native proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel secreted and membrane-bound receptor proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996);U.S. Pat. No. 5,536,637)]. We herein describe the identification andcharacterization of novel polypeptides having significant homology tothe ITI heavy chain, designated in the present application as PRO354polypeptides.

[0036] 10. PRO355

[0037] Cytotoxic or regulatory T cell associated molecule or “CRTAM”protein is structurally related to the immunoglobulin superfamily. TheCRTAM protein should be capable of mediating various immune responses.Antibodies typically bind to CRTAM proteins with high affinity. Zlotnik,A., Faseb, 10(6): A1037, Abr. 216, June 1996. Given the physiologicalimportance of T cell antigens and immune processes in vivo, efforts arecurrently being under taken to identify new, native proteins which areinvolved in immune responses. See also Kennedy et al., U.S. Pat. No.5,686,257 (1997). We describe herein the identification andcharacterization of novel polypeptides which have homology to CRTAM,designated in the present application as PRO355 polypeptides.

[0038] 11. PRO357

[0039] Protein-protein interactions include receptor and antigencomplexes and signaling mechanisms. As more is known about thestructural and functional mechanisms underlying protein-proteininteractions, protein-protein interactions can be more easilymanipulated to regulate the particular result of the protein-proteininteraction. Thus, the underlying mechanisms of protein-proteininteractions are of interest to the scientific and medical community.

[0040] All proteins containing leucine-rich repeats are thought to beinvolved in protein-protein interactions. Leucine-rich repeats are shortsequence motifs present in a number of proteins with diverse functionsand cellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglobular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October. 1994).

[0041] A study has been reported on leucine-rich proteoglycans whichserve as tissue organizers, orienting and ordering collagen fibrilsduring ontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndrome,Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1): 111-116 (July1995), reporting that platelets have leucine rich repeats and Ruoslahti,E. I., et al., WO9110727-A by La Jolla Cancer Research Foundationreporting that decorin binding to transforming growth factorβ hasinvolvement in a treatment for cancer, wound healing and scarring.Related by function to this group of proteins is the insulin like growthfactor (IGF), in that it is useful in wound-healing and associatedtherapies concerned with re-growth of tissue, such as connective tissue,skin and bone; in promoting body growth in humans and animals; and instimulating other growth-related processes. The acid labile subunit(ALS) of IGF is also of interest in that it increases the half-life ofIGF and is part of the IGF complex in vivo.

[0042] Another protein which has been reported to have leucine-richrepeats is the SLIT protein which has been reported to be useful intreating neuro-degenerative diseases such as Alzheimer's disease, nervedamage such as in Parkinson's disease, and for diagnosis of cancer, see,Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by YaleUniversity. Also of interest is LIG-1, a membrane glycoprotein that isexpressed specifically in glial cells in the mouse brain, and hasleucine rich repeats and immunoglobulin-like domains. Suzuki, et al., J.Biol. Chem. (U.S.), 271(37):22522 (1996). Other studies reporting on thebiological functions of proteins having leucine rich repeats include:Tayar, N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70(December. 1996) (gonadotropin receptor involvement); Miura, Y., et al.,Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosisinvolvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,6(4):1125-1133 (October. 1995(kidney disease involvement).

[0043] Efforts are therefore being undertaken by both industry andacademia to identify new proteins having leucine rich repeats to betterunderstand protein-protein interactions. Of particular interest arethose proteins having leucine rich repeats and homology to knownproteins having leucine rich repeats such as the acid labile subunit ofinsulin-like growth factor. Many efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel secreted and membrane-bound proteins having leucine rich repeats.Examples of screening methods and techniques are described in theliterature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

[0044] We describe herein the identification and characterization ofnovel polypeptides having homology to the acid labile subunit ofinsulin-like growth factor, designated in the present application asPRO357 polypeptides.

[0045] 12. PRO715

[0046] Control of cell numbers in mammals is believed to be determined,in part, by a balance between cell proliferation and cell death. Oneform of cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al., Bio/Technology12:487-493 (1994); Steller et al., Science, 267:1445-1449 (1995)].Apoptotic cell death naturally occurs in many physiological processes,including embryonic development and clonal selection in the immunesystem [Itoh et al., Cell, 66:233-243 (1991)]. Decreased levels ofapoptotic cell death have been associated with a variety of pathologicalconditions, including cancer, lupus, and herpes virus infection[Thompson, Science, 267:1456-1462 (1995)]. Increased levels of apoptoticcell death may be associated with a variety of other pathologicalconditions, including AIDS, Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa,cerebellar degeneration, aplastic anemia, myocardial infarction, stroke,reperfusion injury, and toxin-induced liver disease [see, Thompson,supra].

[0047] Apoptotic cell death is typically accompanied by one or morecharacteristic morphological and biochemical changes in cells, such ascondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. A variety of extrinsic and intrinsic signals arebelieved to trigger or induce such morphological and biochemicalcellular changes [Raff, Nature, 356:397400 (1992); Steller, supra; Sachset al., Blood, 82:15 (1993)]. For instance, they can be triggered byhormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)]. Also, someidentified oncogenes such as myc, rel, and EIA, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

[0048] Various molecules, such as tumor necrosis factor-α” (“TNF-α”),tumor necrosis factor-β (“TNF-β” or “lymphotoxin-α”), lymphotoxin-β(“LT-β”), CD30 ligand, CD27 ligand, CD40 ligand, OX40 ligand, 4-1BBligand, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand),and Apo-2 ligand (also referred to as TRAIL) have been identified asmembers of the tumor necrosis factor (“TNF”) family of cytokines [See,e.g., Gruss and Dower, Blood, 85:3378-3404 (1995); Pitti et al., J.Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity, 3:673-682(1995); Browning et al., Cell, 72:847-856 (1993); Armitage et al.Nature, 357-80-82 (1992)]. Among these molecules, TNF-α, TNF-β, CD30ligand, 4-1BB ligand, Apo-1 ligand, and Apo-2 ligand (TRAIL) have beenreported to be involved in apoptotic cell death. Both TNF-α and TNF-βhave been reported to induce apoptotic death in susceptible tumor cells[Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al.,Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-αis involved in post-stimulation apoptosis of CD8-positive T cells [Zhenget al., Nature, 377:348-351 (1995)]. Other investigators have reportedthat CD30 ligand may be involved in deletion of self-reactive T cells inthe thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

[0049] Mutations in the mouse Fas/Apo-1 receptor or ligand genes (calledlpr and gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol. 6:279-289 (1994); Nagata et al.,Science 267:1449-1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].

[0050] Induction of various cellular responses mediated by such TNFfamily cytokines is believed to be initiated by their binding tospecific cell receptors. Two distinct TNF receptors of approximately55-kDa (TNFR1) and 75-kDa (TNFR2) have been identified [Hohman et al.,J. Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Proc. Natl.Acad. Sci., 87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991]and human and mouse cDNAs corresponding to both receptor types have beenisolated and characterized [Loetscher et al., Cell, 61:351 (1990);Schall et al., Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023(1990); Lewis et. al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991);Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991). The TNF familyligands identified to date, with the exception of lymphotoxin-α, aretype II transmembrane proteins, whose C-terminus is extracellular. Incontrast, most receptors in the TNF receptor (TNFR) family identified todate are type I transmembrane proteins. In both the TNF ligand andreceptor families, however, homology identified between family membershas been found mainly in the extracellular domain (“ECD”). Several ofthe TNF family cytokines, including TNF-α, Apo-1 ligand and CD40 ligand,are cleaved proteolytically at the cell surface; the resulting proteinin each case typically forms a homotrimeric molecule that functions as asoluble cytokine. TNF receptor family proteins are also usually cleavedproteolytically to release soluble receptor ECDs that can function asinhibitors of the cognate cytokines.

[0051] Recently, other members of the TNFR family have been identifiedSuch newly identified members of the TNFR family include CAR1, HVEM andosteoprotegerin (OPG) [Brojatsch et al., Cell, 87:845-855 (1996);Montgomery et al., Cell, 87:427436 (1996); Marsters et al., J. Biol.Chem., 272:14029-14032 (1997); Simonet et al., Cell, 89:309-319 (1997)].Unlike other known TNFR-like molecules, Simonet et al., supra, reportthat OPG contains no hydrophobic transmembrane-spanning sequence.

[0052] For a review of the TNF family of cytokines and their receptors,see Gruss and Dower, supra.

[0053] Applicants herein describe the identification andcharacterization of novel polypeptides having homology to members of thetumor necrosis factor family of polypeptides, designated herein asPRO715 polypeptides.

[0054] 13. PRO353

[0055] The complement proteins comprise a large group of serum proteinssome of which act in an enzymatic cascade, producing effector moleculesinvolved in inflammation. The complement proteins are of particularimportance in regulating movement and function of cells involved ininflammation. Given the physiological importance of inflammation andrelated mechanisms in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in inflamation. Wedescribe herein the identification and characterization of novelpolypeptides which have homology to complement proteins, designatedherein as PRO353 polypeptides.

[0056] 14. PRO361

[0057] The mucins comprise a family of glycoproteins which have beenimplicated in carcinogenesis. Mucin and mucin-like proteins are secretedby both normal and transformed cells. Both qualitative and quantitativechanges in mucins have been implicated in various types of cancer. Giventhe medical importance of cancer, efforts are currently being undertaken to identify new, native proteins which may be useful for thediagnosis or treatment of cancer.

[0058] The chitinase proteins comprise a family of which have beenimplicated in pathogenesis responses in plants. Chitinase proteins areproduced by plants and microorganisms and may play a role in the defenseof plants to injury. Given the importance of plant defense mechanisms,efforts are currently being under taken to identify new, native proteinswhich may be useful for modulation of pathogenesis-related responses inplants. We describe herein the identification and characterization ofnovel polypeptides which have homology to mucin and chitinase,designated in the present application as PRO361 polypeptides.

[0059] 15. PRO365

[0060] Polypeptides such as human 2-19 protein may function ascytokines. Cytokines are low molecular weight proteins which function tostimulate or inhibit the differentiation, proliferation or function ofimmune cells. Cytokines often act as intercellular messengers and havemultiple physiological effects. Given the physiological importance ofimmune mechanisms in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in effecting the immunesystem. We describe herein the identification and characterization ofnovel polypeptides which have homology to the human 2-19 protein,designated herein as PRO365 polypeptides.

SUMMARY OF THE INVENTION

[0061] 1. PRO241

[0062] Applicants have identified a cDNA clone that encodes a novelpolypeptide having homology to biglycan protein, wherein the polypeptideis designated in the present application as “PRO241”.

[0063] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO241 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO241polypeptide having amino acid residues 1 to 379 of FIG. 2 (SEQ ID NO:2),or is complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions.

[0064] In another embodiment, the invention provides isolated PRO241polypeptide. In particular, the invention provides isolated nativesequence PRO241 polypeptide, which in one embodiment, includes an aminoencoding nucleic acid sequence comprising residues 1 to 379 of FIG. 2(SEQ ID NO:2). Another embodiment of the present invention is directedto a PRO241 polypeptide lacking the N-terminal signal peptide, whereinthe PRO241 polypeptide comprises about amino acids 16 to 379 of thefull-length PRO241 amino acid sequence (SEQ ID NO:2).

[0065] 2. PRO243

[0066] Applicants have identified a cDNA clone (DNA35917-1207) thatencodes a novel polypeptide, designated in the present application as“PRO243”.

[0067] In one embodiment, the invention provides an isolated nucleicacid molecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a PRO243 polypeptide comprising the sequence of aminoacids 1 or about 24 to 954 of FIG. 4 (SEQ ID NO:7), or (b) thecomplement of the DNA molecule of (a). The sequence identity preferablyis about 85%, more preferably about 90%, most preferably about 95%. Inone aspect, the isolated nucleic acid has at least about 80%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% sequence identity with a polypeptidehaving amino acid residues 1 or about 24 to 954 of FIG. 4 (SEQ ID NO:7).Preferably, the highest degree of sequence identity occurs within thefour (4) conserved cysteine clusters (amino acids 51 to 125; amino acids705 to 761; amino acids 784 to 849; and amino acids 897 to 931) of FIG.4 (SEQ ID NO:7). In a further embodiment, the isolated nucleic acidmolecule comprises DNA encoding a PRO243 polypeptide having amino acidresidues 1 or about 24 to 954 of FIG. 4 (SEQ ID NO:7), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the invention provides anucleic acid of the full length protein of clone DNA35917-1207,deposited with the ATCC under accession number ATCC 209508,alternatively the coding sequence of clone DNA35917-1207, depositedunder accession number ATCC 209508.

[0068] In yet another embodiment, the invention provides isolated PRO243polypeptide. In particular, the invention provides isolated nativesequence PRO243 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or about 24 to 954 of FIG. 4 (SEQ IDNO:7). Native PRO243 polypeptides with or without the native signalsequence (amino acids 1 to 23 in FIG. 4 (SEQ ID NO:7)), and with orwithout the initiating methionine are specifically included.Alternatively, the invention provides a PRO243 polypeptide encoded bythe nucleic acid deposited under accession number ATCC 209508.

[0069] 3. PRO299

[0070] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO299”.

[0071] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO299 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO299polypeptide having amino acid residues 1 to 737 of FIG. 6 (SEQ IDNO:15), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0072] In another embodiment, the invention provides isolated PRO299polypeptide. In particular, the invention provides isolated nativesequence PRO299 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 737 of FIG. 6 (SEQ ID NO:15). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO299 polypeptide.

[0073] 4. PRO323

[0074] Applicants have identified a cDNA clone that encodes a novelpolypeptide having homology to a microsomal dipeptidase protein, whereinthe polypeptide is designated in the present application as “PRO323”.

[0075] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO323 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO323polypeptide having amino acid residues 1 to 433 of FIG. 10 (SEQ IDNO:24), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0076] In another embodiment, the invention provides isolated PRO323polypeptide. In particular, the invention provides isolated nativesequence PRO323 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 433 of FIG. 10 (SEQ ID NO:24).

[0077] 5. PRO327

[0078] Applicants have identified a cDNA clone that encodes a novelpolypeptide having homology to prolactin receptor, wherein thepolypeptide is designated in the present application as “PRO327”.

[0079] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO327 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO327polypeptide having amino acid residues 1 to 422 of FIG. 14 (SEQ IDNO:32), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0080] In another embodiment, the invention provides isolated PRO327polypeptide. In particular, the invention provides isolated nativesequence PRO327 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 422 of FIG. 14 (SEQ ID NO:32).

[0081] 6. PRO233

[0082] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is are designated in the presentapplication as “PRO233”.

[0083] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO233 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO233polypeptide having amino acid residues 1 to 300 of FIG. 16 (SEQ IDNO:37), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0084] In another embodiment, the invention provides isolated PRO233polypeptide. In particular, the invention provides isolated nativesequence PRO233 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 300 of FIG. 16 (SEQ ID NO:37).

[0085] 7. PRO344

[0086] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptides are designated in the presentapplication as “PRO344”.

[0087] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO344 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO344polypeptide having amino acid residues 1 to 243 of FIG. 18 (SEQ IDNO:42), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0088] In another embodiment, the invention provides isolated PRO344polypeptide. In particular, the invention provides isolated nativesequence PRO344 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 243 of FIG. 18 (SEQ ID NO:42).

[0089] 8. PRO347

[0090] Applicants have identified a cDNA clone that encodes a novelpolypeptide having homology to cysteine-rich secretory protein-3,wherein the polypeptide is designated in the present application as“PRO347”.

[0091] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO347 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO347polypeptide having amino acid residues 1 to 455 of FIG. 20 (SEQ IDNO:50), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0092] In another embodiment, the invention provides isolated PRO347polypeptide. In particular, the invention provides isolated nativesequence PRO347 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 455 of FIG. 20 (SEQ ID NO:50).

[0093] 9. PRO354

[0094] Applicants have identified a cDNA clone that encodes a novelpolypeptide having homology to the heavy chain of theinter-alpha-trypsin inhibitor (ITI), wherein the polypeptide isdesignated in the present application as “PRO354”.

[0095] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO354 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO354polypeptide having amino acid residues 1 to 694 of FIG. 22 (SEQ IDNO:55), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0096] In another embodiment, the invention provides isolated PRO354polypeptide. In particular, the invention provides isolated nativesequence PRO354 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 694 of FIG. 22 (SEQ ID NO:55).

[0097] 10. PRO355

[0098] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO355”.

[0099] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO355 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO355polypeptide having amino acid residues 1 to 440 of FIG. 24 (SEQ IDNO:61), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0100] In another embodiment, the invention provides isolated PRO355polypeptide. In particular, the invention provides isolated nativesequence PRO355 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 440 of FIG. 24 (SEQ ID NO:61). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO355 polypeptide.

[0101] 11. PRO357

[0102] Applicants have identified a cDNA clone that encodes a novelpolypeptide having homology to insulin-like growth factor (IGF) acidlabile subunit (ALS), wherein the polypeptide is designated in thepresent application as “PRO357”.

[0103] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO357 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO357polypeptide having amino acid residues 1 through 598 of FIG. 26 (SEQ IDNO:69), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0104] In another embodiment, the invention provides isolated PRO357polypeptide. In particular, the invention provides isolated nativesequence PRO357 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 598 of FIG. 26 (SEQ IDNO:69). An additional embodiment of the present invention is directed toan isolated extracellular domain of a PRO357 polypeptide.

[0105] 12. PRO715

[0106] Applicants have identified cDNA clones that encode novelpolypeptides having homology to tumor necrosis factor familypolypeptides, wherein the polypeptides are designated in the presentapplication as “PRO715”.

[0107] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO715 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO715polypeptide having amino acid residues 1 to 250 of FIG. 28 (SEQ IDNO:76), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0108] In another embodiment, the invention provides isolated PRO715polypeptide. In particular, the invention provides isolated nativesequence PRO715 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 250 of FIG. 28 (SEQ ID NO:76). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO715 polypeptide.

[0109] 13. PRO353

[0110] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptides are designated in the presentapplication as “PRO353”.

[0111] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO353 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO353polypeptide having amino acid residues 1 to 281 of FIG. 30 (SEQ IDNO:78), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions.

[0112] In another embodiment, the invention provides an isolated PRO353polypeptide. In particular, the invention provides isolated nativesequence PRO353 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 281 of FIG. 30 (SEQ ID NO:78).

[0113] 14. PRO361

[0114] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO361”.

[0115] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO361 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO361polypeptide having amino acid residues 1 to 431 of FIG. 32 (SEQ IDNO:83), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. The isolated nucleic acid sequence maycomprise the cDNA insert of the vector deposited on Feb. 5, 1998 as ATCC209621 which includes the nucleotide sequence encoding PRO361.

[0116] In another embodiment, the invention provides isolated PRO361polypeptide. In particular, the invention provides isolated nativesequence PRO361 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 431 of FIG. 32 (SEQ ID NO:83). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO361 polypeptide having amino acids1 to 379 of the amino acids sequence shown in FIG. 32 (SEQ ID NO:83).Optionally, the PRO361 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited on February 5, 1998 as ATCC 209621.

[0117] 15. PRO365

[0118] Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO365”.

[0119] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO365 polypeptide. In oneaspect, the isolated nucleic acid comprises DNA encoding the PRO365polypeptide having amino acid residues 1 to 235 of FIG. 34 (SEQ IDNO:91), or is complementary to such encoding nucleic acid sequence, andremains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another aspect, the isolatednucleic acid comprises DNA encoding the PRO365 polypeptide having aminoacid residues 21 to 235 of FIG. 34 (SEQ ID NO:91), or is complementaryto such encoding nucleic acid sequence, and remains stably bound to itunder at least moderate, and optionally, under high stringencyconditions.

[0120] In another embodiment, the invention provides isolated PRO365polypeptide. In particular, the invention provides isolated nativesequence PRO365 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 235 of FIG. 34 (SEQ ID NO:91). Anadditional embodiment of the present invention is directed to an aminoacid sequence comprising residues 21 to 235 of FIG. 34 (SEQ ID NO:91).

[0121] 16. Additional Embodiments

[0122] In other embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

[0123] In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

[0124] In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

[0125] In yet other embodiments, the invention provides oligonucleotideprobes useful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

[0126] In other embodiments, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes a PROpolypeptide.

[0127] In one aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculeencoding a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

[0128] In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein, the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein or the coding sequence of anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

[0129] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC as disclosed herein, or (b)the complement of the DNA molecule of (a).

[0130] Another aspect the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed PRO polypeptides are contemplated.

[0131] Another embodiment is directed to fragments of a PRO polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of a PROpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO antibody or as antisense oligonucleotideprobes. Such nucleic acid fragments are usually at least about 20nucleotides in length, preferably at least about 30 nucleotides inlength, more preferably at least about 40 nucleotides in length, yetmore preferably at least about 50 nucleotides in length, yet morepreferably at least about 60 nucleotides in length, yet more preferablyat least about 70 nucleotides in length, yet more preferably at leastabout 80 nucleotides in length, yet more preferably at least about 90nucleotides in length, yet more preferably at least about 100nucleotides in length, yet more preferably at least about 110nucleotides in length, yet more preferably at least about 120nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleolides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PROpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO polypeptide-encoding nucleotide sequence withother known nucleotide sequences using any of a number of well knownsequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

[0132] In another embodiment, the invention provides isolated PROpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0133] In a certain aspect, the invention concerns an isolated PROpolypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 9 8% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

[0134] In a further aspect, the invention concerns an isolated PROpolypeptide comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC as disclosed herein.

[0135] In a further aspect, the invention concerns an isolated PROpolypeptide comprising an amino acid sequence scoring at least about 80%positives, preferably at least about 81% positives, more preferably atleast about 82% positives, yet more preferably at least about 83%positives, yet more preferably at least about 84% positives, yet morepreferably at least about 85% positives, yet more preferably at leastabout 86% positives, yet more preferably at least about 87% positives,yet more preferably at least about 88% positives, yet more preferably atleast about 89% positives, yet more preferably at least about 90%positives, yet more preferably at least about 91% positives, yet morepreferably at least about 92% positives, yet more preferably at leastabout 93% positives, yet more preferably at least about 94% positives,yet more preferably at least about 95% positives, yet more preferably atleast about 96% positives, yet more preferably at least about 97%positives, yet more preferably at least about 98% positives and yet morepreferably at least about 99% positives when compared with the aminoacid sequence of a PRO polypeptide having a full-length amino acidsequence as disclosed herein, an amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneprotein, with or without the signal peptide, as disclosed herein or anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein.

[0136] In a specific aspect, the invention provides an isolated PROpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as hereinbefore described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO polypeptide and recovering the PRO polypeptidefrom the cell culture.

[0137] Another aspect the invention provides an isolated PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

[0138] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

[0139] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists to a PRO polypeptide which comprisecontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

[0140] In a still further embodiment, the invention concerns acomposition of matter comprising a PRO polypeptide, or an agonist orantagonist of a PRO polypeptide as herein described, or an anti-PROantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

[0141] Another embodiment of the present invention is directed to theuse of a PRO polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-PRO antibody, for the preparation ofa medicament useful in the treatment of a condition which is responsiveto the PRO polypeptide, an agonist or antagonist thereof or an anti-PROantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0142]FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a nativesequence PRO241 cDNA, wherein SEQ ID NO:1 is a clone designated hereinas “DNA34392-1170”.

[0143]FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived fromthe coding sequence of SEQ ID NO:1 shown in FIG. 1.

[0144]FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a nativesequence PRO243 cDNA, wherein SEQ ID NO:6 is a clone designated hereinas “DNA35917-1207”.

[0145]FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived fromthe coding sequence of SEQ ID NO:6 shown in FIG. 3.

[0146]FIG. 5 shows a nucleotide sequence (SEQ ID NO:14) of a nativesequence PRO299 cDNA, wherein SEQ ID NO:14 is a clone designated hereinas “DNA39976-1215”.

[0147]FIG. 6 shows the amino acid sequence (SEQ ID NO:15) derived fromthe coding sequence of SEQ ID NO:14 shown in FIG. 5.

[0148]FIG. 7 shows a nucleotide sequence designated herein as DNA28847(SEQ ID NO:18).

[0149]FIG. 8 shows a nucleotide sequence designated herein as DNA35877(SEQ ID NO:19).

[0150]FIG. 9 shows a nucleotide sequence (SEQ ID NO:23) of a nativesequence PRO323 cDNA, wherein SEQ ID NO:23 is a clone designated hereinas “DNA35595-1228”.

[0151]FIG. 10 shows the amino acid sequence (SEQ ID NO:24) derived fromthe coding sequence of SEQ ID NO:23 shown in FIG. 9.

[0152]FIG. 11 shows a single-stranded nucleotide sequence (SEQ ID NO:29)containing the nucleotide sequence (nucleotides 79-1416) of a chimericfusion protein between a PRO323-derived polypeptide and a portion of anIgG constant domain, wherein the chimeric fusion protein is designatedherein as “PRO454”. The single-stranded nucleotide sequence (SEQ IDNO:29) encoding the PRO323/IgG fusion protein (PRO454) is designatedherein as “DNA35872”.

[0153]FIG. 12 shows the amino acid sequence (SEQ ID NO:30) derived fromnucleotides 79-1416 of the nucleotide sequence shown in FIG. 11. Thejunction in the PRO454 amino acid sequence between the PRO323-derivedsequences and the IgG-derived sequences appears between amino acids415-416 in the figure.

[0154]FIG. 13 shows a nucleotide sequence (SEQ ID NO:31) of a nativesequence PRO327 cDNA, wherein SEQ ID NO:31 is a clone designated hereinas “DNA38113-1230”.

[0155]FIG. 14 shows the amino acid sequence (SEQ ID NO:32) derived fromthe coding sequence of SEQ ID NO:31 shown in FIG. 13.

[0156]FIG. 15 shows a nucleotide sequence (SEQ ID NO:36) of a nativesequence PRO233 cDNA, wherein SEQ ID NO:36 is a clone designated hereinas “DNA34436-1238”.

[0157]FIG. 16 shows the amino acid sequence (SEQ ID NO:37) derived fromthe coding sequence of SEQ ID NO:36 shown in FIG. 15.

[0158]FIG. 17 shows a nucleotide sequence (SEQ ID NO:41) of a nativesequence PRO344 cDNA, wherein SEQ ID NO:41 is a clone designated hereinas “DNA40592-1242”.

[0159]FIG. 18 shows the amino acid sequence (SEQ ID NO:42) derived fromthe coding sequence of SEQ ID NO:41 shown in FIG. 17.

[0160]FIG. 19 shows a nucleotide sequence (SEQ ID NO:49) of a nativesequence PRO347 cDNA, wherein SEQ ID NO:49 is a clone designated hereinas “DNA44176-1244”.

[0161]FIG. 20 shows the amino acid sequence (SEQ ID NO:50) derived fromthe coding sequence of SEQ ID NO:49 shown in FIG. 19.

[0162]FIG. 21 shows a nucleotide sequence (SEQ ID NO:54) of a nativesequence PRO354 cDNA, wherein SEQ ID NO:54 is a clone designated hereinas “DNA44192-1246”.

[0163]FIG. 22 shows the amino acid sequence (SEQ ID NO:55) derived fromthe coding sequence of SEQ ID NO:54 shown in FIG. 21.

[0164]FIG. 23 shows a nucleotide sequence (SEQ ID NO:60) of a nativesequence PRO355 cDNA, wherein SEQ ID NO:60 is a clone designated hereinas “DNA39518-1247”.

[0165]FIG. 24 shows the amino acid sequence (SEQ ID NO:61) derived fromthe coding sequence of SEQ ID NO:60 shown in FIG. 23.

[0166]FIG. 25 shows a nucleotide sequence (SEQ ID NO:68) of a nativesequence PRO357 cDNA, wherein SEQ ID NO:68 is a clone designated hereinas “DNA44804-1248”.

[0167]FIG. 26 shows the amino acid sequence (SEQ ID NO:69) derived fromthe coding sequence of SEQ ID NO:68 shown in FIG. 25.

[0168]FIG. 27 shows a nucleotide sequence (SEQ ID NO:75) of a nativesequence PRO715 cDNA, wherein SEQ ID NO:75 is a clone designated hereinas “DNA52722-1229”.

[0169]FIG. 28 shows the amino acid sequence (SEQ ID NO:76) derived fromthe coding sequence of SEQ ID NO:75 shown in FIG. 27.

[0170]FIG. 29 shows a nucleotide sequence (SEQ ID NO:77) of a nativesequence PRO353 cDNA, wherein SEQ ID NO:77 is a clone designated hereinas “DNA41234-1242”.

[0171]FIG. 30 shows the amino acid sequence (SEQ ID NO:78) derived fromthe coding sequence of SEQ ID NO:77 shown in FIG. 29.

[0172]FIG. 31 shows a nucleotide sequence (SEQ ID NO:82) of a nativesequence PRO361 cDNA, wherein SEQ ID NO:82 is a clone designated hereinas “DNA45410-1250”.

[0173]FIG. 32 shows the amino acid sequence (SEQ ID NO:83) derived fromthe coding sequence of SEQ ID NO:82 shown in FIG. 31.

[0174]FIG. 33 shows a nucleotide sequence (SEQ ID NO:90) of a nativesequence PRO365 cDNA, wherein SEQ ID NO:90 is a clone designated hereinas “DNA46777-1253”.

[0175]FIG. 34 shows the amino acid sequence (SEQ ID NO:91) derived fromthe coding sequence of SEQ ID NO:90 shown in FIG. 33.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0176] The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

[0177] A “native sequence PRO polypeptide” comprises a polypeptidehaving the same amino acid sequence as the corresponding PRO polypeptidederived from nature. Such native sequence PRO polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific PRO polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

[0178] The PRO polypeptide “extracellular domain” or “ECD” refers to aform of the PRO polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECDwill have less than 1% of such transmembrane and/or cytoplasmic domainsand preferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the PROpolypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are contemplated by thepresent invention.

[0179] The approximate location of the “signal peptides” of the variousPRO polypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:46834690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

[0180] “PRO polypeptide variant” means an active PRO polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence PRO polypeptide sequence asdisclosed herein, a PRO polypeptide sequence lacking the signal peptideas disclosed herein, an extracellular domain of a PRO polypeptide, withor without the signal peptide, as disclosed herein or any other fragmentof a full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often a t least about 300amino acids in length, or more.

[0181] “Percent (%) amino acid sequence identity” with respect to thePRO polypeptide sequence s identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific PRO polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Forpurposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2,wherein the complete source code for the ALIGN-2 program is provided inTable 1 below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in Table 1 belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1 below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

[0182] In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0183] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. As examples of % amino acid sequenceidentity calculations using this method, Tables 2 and 3 demonstrate howto calculate the % amino acid sequence identity of the amino acidsequence designated “Comparison Protein” to the amino acid sequencedesignated “PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

[0184] Unless specifically stated otherwise, all % amino acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

[0185] Percent amino acid sequence identity may also be determined usingthe sequence comparison program NCBI-BLAST2 (Altschul et al., NucleicAcids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparisonprogram may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2uses several search parameters, wherein all of those search parametersare set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0186] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0187] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0188] “PRO variant polynucleotide” or “PRO variant nucleic acidsequence” means a nucleic acid molecule which encodes an active PROpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Ordinarily, a PRO variant polynucleotide will have atleast about 80% nucleic acid sequence identity, more preferably at leastabout 81% nucleic acid sequence identity, more preferably at least about82% nucleic acid sequence identity, more preferably at least about 83%nucleic acid sequence identity, more preferably at least about 84%nucleic acid sequence identity, more preferably at least about 85%nucleic acid sequence identity, more preferably at least about 86%nucleic acid sequence identity, more preferably at least about 87%nucleic acid sequence identity, more preferably at least about 88%nucleic acid sequence identity, more preferably at least about 89%nucleic acid sequence identity, more preferably at least about 90%nucleic acid sequence identity, more preferably at least about 91%nucleic acid sequence identity, more preferably at least about 92%nucleic acid sequence identity, more preferably at least about 93%nucleic acid sequence identity, more preferably at least about 94%nucleic acid sequence identity, more preferably at least about 95%nucleic acid sequence identity, more preferably at least about 96%nucleic acid sequence identity, more preferably at least about 97%nucleic acid sequence identity, more preferably at least about 98%nucleic acid sequence identity and yet more preferably at least about99% nucleic acid sequence identity with a nucleic acid sequence encodinga full-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

[0189] Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more. “Percent (%) nucleic acid sequenceidentity” with respect to PRO-encoding nucleic acid sequences identifiedherein is defined as the percentage of nucleotides in a candidatesequence that are identical with the nucleotides in the PRO nucleic acidsequence of interest, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposesherein, however, % nucleic acid sequence identity values are generatedusing the sequence comparison computer program ALIGN-2, wherein thecomplete source code for the ALIGN-2 program is provided in Table 1below. The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc. and the source code shown in Table 1 below has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. or may be compiled from thesource code provided in Table 1 below. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

[0190] In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0191] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

[0192] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

[0193] Percent nucleic acid sequence identity may also be determinedusing the sequence comparison program NCBI-BLAST2 (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequencecomparison program may be downloaded from http://www.ncbi.nlm.nih.gov.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0194] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0195] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0196] In other embodiments, PRO variant polynucleotides are nucleicacid molecules that encode an active PRO polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

[0197] The term “positives”, in the context of sequence comparisonperformed as described above, includes residues in the sequencescompared that are not identical but have similar properties (e.g. as aresult of conservative substitutions, see Table 6 below). For purposesherein, the % value of positives is determined by dividing (a) thenumber of amino acid residues scoring a positive value between the PROpolypeptide amino acid sequence of interest having a sequence derivedfrom the native PRO polypeptide sequence and the comparison amino acidsequence of interest (i.e., the amino acid sequence against which thePRO polypeptide sequence is being compared) as determined in theBLOSUM62 matrix of WU-BLAST-2 by (b) the total number of amino acidresidues of the PRO polypeptide of interest.

[0198] Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

[0199] For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2,the % value of positives of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% positives to, with, or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction X/Y

[0200] where X is the number of amino acid residues scoring a positivevalue as defined above by the sequence alignment program ALIGN-2 orNCBI-BLAST2 in that program's alignment of A and B, and where Y is thetotal number of amino acid residues in B. It will be appreciated thatwhere the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % positives of A to B will not equal the %positives of B to A.

[0201] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0202] An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

[0203] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0204] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0205] The term “antibody” is used in the broadest sense andspecifically covers, for example, single anti-PRO monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies), anti-PROantibody compositions with polyepitopic specificity, single chainanti-PRO antibodies, and fragments of anti-PRO antibodies (see below).The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts.

[0206] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology. Wiley IntersciencePublishers, (1995).

[0207] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0. 1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55 ° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0208] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0209] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

[0210] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

[0211] “Active” or “activity” for the purposes herein refers to form(s)of a PRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

[0212] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

[0213] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

[0214] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

[0215] “Intermittent” administration is treatment that is notconsecutively done without interruption, but rather is cyclic in nature.

[0216] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0217] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0218] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0219] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0220] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab”fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen.

[0221] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0222] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fabfragments differ from Fab′ fragments by the addition of a few residuesat the carboxy terminus of the heavy chain CH1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

[0223] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains.

[0224] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0225] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0226] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0227] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0228] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

[0229] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0230] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

[0231] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons. TABLE 1 /* * * C-C increased from 12 to 15 * Zis average of EQ * B is average of ND * match with stop is _M; stop-stop0; J (joker) match = 0 */ #define _M −8 /* value of a match with a stop*/ int   _day[26][26] = { /*    A B C D E F G H I J K L M N O P Q R S TU V W X Y Z */ /* A */ {2, 0, −2, 0, 0, 4, 1, −1, −1, 0, −1, −2, −1, 0,_M, 1, 0, −2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ {0, 3, −4, 3, 2, −5, 0,1, −2, 0, 0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0, −3, 1}, /* C*/ {−2, −4, 15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4, _M, −3, −5,−4, 0, −2, 0, −2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3, −6, 1, 1, −2,0, 0, −4, −3, 2, _M, −1, 2, −1, 0, 0. 0, −2, −7, 0, −4, 2}, /* E */ {0,2, −5, 3, 4, −5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2, −1, 0, 0, 0, −2,−7, 0, −4, 3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2, 1, 0, −5, 2, 0,−4, _M, −5, −5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */ {1, 0, −3, 1,0, −5, 5, −2, −3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0, 0, −1, −7, 0,−5, 0}, /* H */ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2, −2, 2, M, 0,3, 2, −1, −1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2, −2, −2, 1, −3,−2, 5, 0, −2, 2, 2, −2, M, −2, −2, −2, −1, 0, 0, 4, −5, 0, −1, −2}, /* J*/ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0, 5, −3, 0, 1,_M, −1, 1, 3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3, −6, −4, −3,2, −4, −2, 2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3, −1, 0, 2, −2, 0, −1,−2}, /* M */ {−1, −2, −5 , −3, −2, 0, −3, −2, 2, 0, 0, 4, 6, −2, _M, −2,−1, 0, −2, −1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2, 1, −4, 0, 2,−2, 0, 1, −3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2, 1}, /* O */{_M, _M, _M, _M, _M, _M, _M, _M, _M, _M, _M, _M, _M, _M, 0, _M, _M, _M,_M, _M, _M, _M, _M, _M, _M, _M}, /* P */ {1, −1, −3, −1, −1, −5, −1, 0,−2, 0, −1, −3, −2, −1, _M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /* Q */{0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1, −1, 0,−2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0, 3, −3,0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ {1, 0, 0, 0, 0,−3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0, −3,0}, /* T */ {1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0, −1,−1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2 , −2,−2, −1, −1, −2, 4, 0, −2, 2, 2, −2, _M, −1, −2, −2, −1, 0, 0, 4, −6, 0,−2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4, −4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ {0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1, _M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4} }; /* */ #include<stdio.h> #include <ctype.h> #define MAXJMP 16 /* max jumps in a diag */#define MAXGAP 24 /* don't continue to penalize gaps larger than this */#define JMPS 1024 /* max jmps in an path *f #define MX 4 /* save ifthere's at least MX-1 bases since last jmp */ #define DMAT 3 /* value ofmatching bases */ #define DMIS 0 /* penalty for mismatched bases */#detine DINS0 8 /* penalty for a gap */ #define DINS1 1 /* penalty perbase */ #define PINS0 8 /* penalty for a gap */ #define PINS1 4 /*penalty per residue */ struct jmp { short n[MAXJMP]; /* size of jmp (negfor dely) */ unsigned short x[MAXJMP]; /* base no. of jmp in seq x */ };/* limits seq to 2^ 16 − 1 */ struct diag { int score: /* score at lastjmp */ long offset; /* offset of prev block */ short ijmp; /* currentjmp index */ struct jmp jp; /* list of jmps */ }; struct path { int spc;/* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */int x[JMPS]; /* loc of jmp (last elem before gap) */ }; char *ofile; /*output file name */ char *namex[2]; /* seq names: getseqs() */ char*prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqs() */int dmax; /* best diag: nw() */ int dmax0; /* final diag */ int dna; /*set if dna: main() */ int endgaps; /* set if penalizing end gaps */ intgapx, gapy; /* total gaps in seqs */ int len0, len1; /* seq lens */ intngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw() */int *xbm; /* bitmap for matching */ long offset; /* current offset injmp file */ struct diag *dx; /* holds diagonals */ struct path pp[2]; /*holds path for seqs */ char *calloc(), *malloc() *index() *strcpy();char *getseq(), *g_calloc(); /* Needleman-Wunsch alignment program * *usage: progs file1 file2 * where file1 and file2 are two dna or twoprotein sequences. * The sequences can be in upper- or lower-case an maycontain ambiguity * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored * Max file length is 65535 (limited by unsigned short x in thejmp struct) * A sequence with ⅓ or more of its elements ACGTU is assumedto be DNA * Output is in the file “align.out” * * The program may createa tmp file in /tmp to hold info about traceback. * Original versiondeveloped under BSD 4.3 on a wax 8650 */ #include “nw.h” #include“day.h” static _dbval[26] = { 1, 14, 2, 13, 0, 0, 4, 11, 0, 0, 12, 0, 3,15, 0, 0, 0, 5, 6, 8, 8, 7, 9, 0, 10, 0 }; static _bval[26] = { 1,2|(1<<(‘D’−‘A’))|(1 <<(‘N’−‘A’)), 4, 8, 16, 32, 64, 128, 256, 0xFFFFFFF,1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16, 1<<17, 1<<18, 1<<19,1<<20, 1<<21, 1<<22, 1<<23, 1<<24, 1<<25|(1<<(‘E’−‘A’))|(1<<(‘Q’−‘A’))}; main(ac, av) main int ac; char *av[]; { prog = av[0]; if(ac != 3) {fprintf(stderr, “usage: %s file1 file2\n”, prog); fprintf(stderr, “wherefile1 and file2 are two dna or two protein sequences.\n”);fprintf(stderr, “The sequences can be in upper- or lower-case\n”);fprintf(stderr, “Any lines beginning with ‘;’ or , ‘<’ are ignored\n”);fprintf(stderr, “Output is in the file \“align.out\*\n”); exit(1); }namex[0] = av[1]; namex[1] = av[2]; seqx[0] = getseq(namex[0], &len0);seqx[1] = getseq(namex[1], &len1); xbm = (dna)? _dbval : _pbval; endgaps= 0; /* 1 to penalize endgaps */ ofile = “align.out”; /* output file */nw(); /* fill in the matrix, get the possible jmps */ readjmps(); /* getthe actual jmps */ print(); /* print stats, alignment */ cleanup(0); /*unlink any tmp files */ } /* do the alignment, return best score:main() * dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983 *pro: PAM 250 values * When scores are equal, we prefer mismatches to anygap, prefer * a new gap to extending an ongoing gap, and prefer a gap inseqx * to a gap in seq y. */ nw() nw { char *px, *py; /* seqs and ptrs*/ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keeptrack of delx */ int *tmp; /* for swapping row0, row1 */ int mis; /*score for each type */ int ins0, ins1; /* insertion penalties */register id; /* diagonal index */ register ij; /* jmp index */ register*col0, *col1; /* score for curr, last row */ register xx, yy; /* indexinto seqs */ dx = (struct diag *)g_calloc(“to get diags”, len0 + len1 +1, sizeof(struct diag)); ndely = (int *)g_calloc(“to get ndely”, len1 +1, sizeof(int)); dely = (int *)g_calloc(“to get dely”, len1 + 1,sizeof(int)); col0 = (int *)g_calloc(“to get col0”, len1 + 1,sizeof(int)); col1 = (int *)g_calloc(“to get col1”, len1 + 1,sizeof(int)); ins0 = (dna)? DINS0 : PINS0; ins1 = (dna)? DINS1 : PINS1;smax = −10000; if (endgaps) { for (col0[0] = dely[0] = −ins0, yy = 1; yy<= len1; yy++) { col0[yy] = dely[yy] = col0[yy−1] − ins1; ndely[yy] =yy; } col0[0] = 0; /* Waterman Bull Math Biol 84 */ } else for(yy = 1;yy <= len1; yy++) dely[yy] = −ins0; /* fill in match matrix */ for (px =seqx[0], xx = 1; xx <= len0; px++, xx++) { /* initialize first entry incol */ if (endgaps) { if(xx == 1) col1[0] = delx = −(ins0+ins1); elsecol1[O] = delx = col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx= −ins0; ndelx = 0; } for(py = seqx[1], yy = 1; yy <= len1; py++, yy++){ mis = col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT :DMIS; else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in xseq; * favor new del over ongong del * ignore MAXGAP if weightingendgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq; * favor new del overongong del */ if (endgaps || ndelx < MAXGAP) { if (col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if(col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; were favoring * mis over any del and delx over dely */ id= xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy]) col1[yy] =mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij = dx[id].ijmp; if(dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx > dx[id].jp.x[ij]+MX)|| mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = ndelx;dx[id].jp.x[ij] = xx; dx[id].score = delx; } else { col1[yy] = dely[yy];ij = dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndely[yy] >= MAXJMP &&xx > dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++;if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset= offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = −ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score =dely[yy]; } if (xx == len0 && yy < len1) { /* last col */ if (endgaps)col1[yy] −= ins0 + ins1*(len1−yy); if (col1[yy] > smax) { smax =col1[yy]; dmax = id; } } } if (endgaps && xx < len0) col1[yy−1] −=ins0+ins1*(len0−xx); if(col1[yy−1] > smax) { smax = col1[yy−1]; dmax =id; } tmp = col0; col0 = col1; col1 = tmp; } (void) free((char *)ndely);(void) free((char *)dely); (void) free((char *)col0); (void) free((char*)col1); } /* * * print0 -- only routine visible outside this module * *static: * getmat() -- trace back best path, count matches: print() *pr_align() -- print alignment of described in array p[]: print() *dumpblock() -- dump a block of lines with numbers, stars: pr_align() *nums() -- put out a number line: dumpblock() * putline() -- put out aline (name, [num], seq, num]): dumpblock() * stars() -- put a line ofstars: dumpblock() * stripname() -- strip any path and prefix from aseqname */ #include “nw.h” #define SPC 3 #define P_LINE 256 /* maximumoutput line */ #define P_SPC 3 /* space between name or num and seq */extern _day [26][26]; int olen; /* set output line length */ FILE *fx;/* output file */ print() print { int lx, ly, firstgap, lastgap; /*overlap */ if ((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “%s:can't write %s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<firstsequence: %s (length = %d)\n”, namex[0], len0); fprintf(fx, “<secondsequence: %s (length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly= len1; firstgap = lastgap = 0; if (dmax < len1 − 1) {  /* leading gapin x */ pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } elseif (dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap =dmax − (len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) {/* trailinggap in x */ lastgap = len0 − dmax0 − 1; lx −= lastgap; } else if(dmax0 > len0 − 1) {/* trailing gap in y */ lastgap = dmax0 − (len0 −1); ly −= lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(): }/* * trace back the best path, count matches */ static getmat(lx, ly,firstgap, lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ intfirstgap, lastgap; /* leading trailing overlap */ { int nm, i0, i1,siz0, siz1; char outx[32]; double pct; register n0, n1; register char*p0, *p1; /* get total matches, score */ i0 = i1 = siz0 = siz1 = 0; p0 =seqx[0] + pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while (*p0 && *p1) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else {if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if(n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if(n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++; p1++;} } /* pct homology: * if penalizing endgaps, base is the shorter seq *else, knock off overhangs and take shorter core */ if (endgaps) lx =(len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx); if(gapx) { (void) sprintf(outx, “(%d %s%s”, ngapx, (dna)?“base”:“residue”, (ngapx == 1)? “”:“s”); fprintf(fx, “%s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “(%d %s%s)”, ngapy, (dna)? “base”: “residue”, (ngapy ==1)? “”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “\n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /* * print alignment of described in struct path pp[] */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2; i++) { nn = stripname(namex[i]); if(nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) { for (i =more = 0; i < 2; i++) { /* * do we have more of this sequence? */ if(!*ps[i]) continue; more++; if (pp[i].spc) {  /* leading space */*po[i]++ =‘’; pp[i].spc−− } else if (siz[i]) {  /* in a gap */ *po[i] ++= ‘−’; siz[i]−−; } else { /* we're putting a seq element */ *po[i] =*ps[i]; if (islower(*ps[i])) *p[i] = toupper(*ps[i]); po[i]++; ps[i]++;/* * are we at next gap for this seq? */ if (ni[i] == pp[i].x[ij[i]]) {/* * we need to merge all gaps * at this location */ siz[i] =pp[i].n[ij[i]++]; while (ni[i] == pp[i].x[ij[i]]) siz[i] +=pp[i].n[ij[i]++]; } ni[i]++; } } if(++nn == olen || !more && nn){dumpblock(); for(i = 0; 1 < 2; i++) po[i] = out[i]; nn = 0; } } } /* *dump a block of lines, including numbers, stars: pr_align() */ staticdumpblock() dumpblock { register i; for (i = 0; i < 2; i++) *po[i]−− =‘\0’; (void) putc(‘\n’, fx); for (i = 0; i < 2; i++) { if (*out[i] &&(*out[i] != ‘’ || *(po[i]) != ‘’)) { if(i == 0) nums(i); if (i == 0 &&*out[1]) stars(); putline(i); if (i == 0 && *out[1]) fprintf(fx, star);if(i == 1) nums(i); } } } /* * put out a number line: dumpblock() */static nums(ix) nums int ix; /* index in out[] holding seq line */ {char nline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn= nline, i = 0; i < lmax + P_SPC; i++, pn++) *pn = ‘’; for (i = nc[ix],py = out[ix]; *py; py++, pn++) { if (*py == ‘’ || *py ==‘−’) *pn = ‘’;else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i;for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘−’;} else *pn = ‘’; i++; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn;pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx); } /* * put out a line(name, [num], seq, [num]): dumpblock() */ static putline(ix) putline intix; { int i; register char *px; for (px = namex[ix], i = 0; *p && *px !=‘:’; px++, i++) (void) putc(*px, fx); for (; i < lmax+P_SPC; i++) (void)putc(‘’, fx); /* these count from 1: * ni[] is current element(from 1) * nc[] is number at start of current line */ for (px = out[ix];*px; px++) (void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx); } /* * puta line of stars (seqs always in out[0], out[1]): dumpblock() */ staticstars() stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] ||(*out[0] == ‘’ && *(p0[0]) == ‘’) ||  !*out[1] || (*out[1] == ‘’ &&*(p0[1]) == ‘’)) return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ =‘’; for (p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if(isalpha(*p0) && isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx =‘*’; nm ++; } else if (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’;else cx = ‘’; } else cx = ‘’; *p++ = cx; } *px++ = ‘\n’; *px = ‘\0’; }/* * strip path or prefix from pn, return len: pr_align() */ staticstripname(pn) stripname char *pn; /* file name (may be path) */ {register char *px, *py; py = 0; for (px = pn; *px; px++) if (*px == ‘’)py = px + 1; if (py) (void) strcpy(pn, py); return(strlen(pn)); } /* *cleanup() -- cleanup any tmp file * getseq() -- read in seq, set dna,len, maxlen * g_calloc() -- calloc() with error checkin * readjmps() --get the good jmps, from tmp file if necessary * writejmps() -- write afilled array of jmps to a tmp file: nw() */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup(); /* cleanup tmp file */ long lseek(); /* *remove any tmp file if we blow */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /* * read, return ptr to seq, set dna,len, maxlen * skip lines starting with ‘;’, ‘<’, or ‘>’ * seq in upperor lower case */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if(*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s: malloc() failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; py = pseq + 4; *len = tlen:rewind(fp); while (fgets(line, 1024, fp)) { if(*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) { if(isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ = toupper(*px);if (index(“ATGCU”, *(py−1))) natgc++; } } *py++ = ‘\0’; *py = ‘\0’;(void) fclose(fp); dna = natgc > (tlen/3); return (pseq+4); } char *g_calloc(msg, nx, sz) g_calloc char *msg; /* program, calling routine */int nx, sz; /* number and size of elements */ { char *px, *calloc(); if((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if (*msg) {fprintf(stderr, “%s: g_calloc() failed %s (n=%d, sz=%d)\n”, prog, msg,nx, sz); exit(1); } } return(px); } /* * get final jmps from dx[] or tmpfile, set pp[], reset dmax: main() */ readjmps() readjmps { int fd = −1int siz, i0, i1; register i, j, xx; if (fj) { (void) fclose(fj): if ((fd= open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr, “%s: can't open()%s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1 = 0, dmax0 = dmax,xx = len0; ; i++) { while (1) { for (j = dx[dmax].ijmp; j > = 0 &&dx[dmax].jp.x[j] > = xx; j−−) if (j < 0 && dx[dmax].offset && fj) {(void) lseek(fd, dx[dmax].offset, 0); (void) read(fd, (char*)&dx[dmax].jp, sizeof(struct jmp)); (void) read(fd, (char*)&dx[dmax].offset, sizeof(dx[dmax].offset)); dx[dmax].ijmp = MAXJMP−1;} else break; } if (i >= JMPS) { fprintf(stderr, “%s: too many gaps inalignment\n”, prog); cleanup(1); } if(j >= 0) { siz = dx[dmax].jp.n[j];xx = dx[dmax].jp.x[j]; dmax += siz; if (siz < 0) { /* gap in second seq*/ pp[1].n[i1] = −siz; xx += siz; /* id = xx − yy + len1 − 1 */pp[1].x[i1] = xx − dmax + len1 − 1; gapy++; ngapy −= siz; /* ignoreMAXGAP when doing endgaps */ siz = (−siz < MAXGAP || endgaps)? −siz :MAXGAP; i1++; } else if (siz > 0) { /* gap in first seq */ pp[0].n[i0] =siz; pp[0].x[i0] = xx; gapx++; ngapx += siz; /* ignore MAXGAP when doingendgaps */ siz = (siz < MAXGAP || endgaps)? siz : MAXGAP; i0++; } } elsebreak; } /* reverse the order of jmps */ for (j = 0, i0−−; j < i0; j++,i0−−) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i =pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i; { for (j = 0,i1−−; j < i1; j++, i1−−) { i = pp[1].n[j]; pp[1].n[j] = pp[1].n[i1];pp[1].n[i1] = i; i = pp[1].x[j]; pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] =i; } if (fd >= 0) (void) close(fd); if (fj) { (void) unlink(jname); fj =0; offset = 0; } } /* * write a filled jmp struct offset of the prev one(if any): nw() */ writejmps(ix) writejmps int ix; { char *mktemp(); if(!fj) { if (mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp()%s\n”, prog, jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

[0232] TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein

[0233] (the number of identically matching amino acid residues betweenthe two polypeptide sequences as determined by ALIGN-2) divided by (thetotal number of amino acid residues of the PRO polypeptide)=

[0234] 5 divided by 15=33.3% TABLE 3 PRO XXXXXXXXXX (Length = 10 aminoacids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein

[0235] (the number of identically matching amino acid residues betweenthe two polypeptide sequences as determined by ALIGN-2) divided by (thetotal number of amino acid residues of the PRO polypeptide)=

[0236] 5 divided by 10=50% TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA

[0237] (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence)=

[0238] 6 divided by 14=42.9% TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) 

[0239] (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence)=

[0240] 4 divided by 12=33.3%

[0241] II Compositions and Methods of the Invention

[0242] A. Full-Length PRO Polypeptides

[0243] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO polypeptides. In particular, cDNAs encoding variousPRO polypeptides have been identified and isolated, as disclosed infurther detail in the Examples below. It is noted that proteins producedin separate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

[0244] As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

[0245] 1. Full-length PRO241 Polypeptides

[0246] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO241. In particular, Applicants have identified andisolated cDNA encoding a PRO241 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that portions of the PRO241polypeptide have certain homology with the various biglycan proteins.Accordingly, it is presently believed that PRO241 polypeptide disclosedin the present application is a newly identified biglycan homologpolypeptide and may possess activity typical of biglycan proteins.

[0247] 2. Full-length PRO243 Polypeptides

[0248] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO243. In particular, Applicants have identified andisolated cDNA encoding a PRO243 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST, BLAST-2 and FastA sequencealignment computer programs, Applicants found that a full-length nativesequence PRO243 (shown in FIG. 4 and SEQ ID NO:7) has certain amino acidsequence identity with African clawed frog and Xenopus chordin andcertain homology with rat chordin. Accordingly, it is presently believedthat PRO243 disclosed in the present application is a newly identifiedmember of the chordin protein family and may possess ability toinfluence notochord and muscle formation by the dorsalization of themesoderm.

[0249] 3. Full-length PRO299

[0250] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO299. In particular, Applicants have identified andisolated cDNA encoding a PRO299 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO299polypeptide have certain homology with the notch protein. Accordingly,it is presently believed that PRO299 polypeptide disclosed in thepresent application is a newly identified member of the notch proteinfamily and possesses signaling properties typical of the notch proteinfamily.

[0251] 4. Full-length PRO323 Polypeptides

[0252] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO323. In particular, Applicants have identified andisolated cDNA encoding a PRO323 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO323polypeptide have certain homology with various dipeptidase proteins.Accordingly, it is presently believed that PRO323 polypeptide disclosedin the present application is a newly identified dipeptidase homologthat has dipeptidase activity

[0253] 5. Full-length PRO327 Polypeptides

[0254] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO327. In particular, Applicants have identified andisolated cDNA encoding a PRO327 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that portions of the PRO327polypeptide have certain homology with various prolactin receptorproteins. Accordingly, it is presently believed that PRO327 polypeptidedisclosed in the present application is a newly identified prolactinreceptor homolog and has activity typical of a prolactin receptorprotein.

[0255] 6. Full-length PRO233 Polypeptides

[0256] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO233. In particular, Applicants have identified andisolated cDNA encoding a PRO233 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO233polypeptide have certain homology with various reductase proteins.Applicants have also found that the DNA encoding the PRO233 polypeptidehas significant homology with proteins from Caenorhabditis elegans.Accordingly, it is presently believed that PRO233 polypeptide disclosedin the present application is a newly identified member of the reductasefamily and possesses the ability to effect the redox state of a celltypical of the reductase family.

[0257] 7. Full-length PRO344 Polypeptides

[0258] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO344. In particular, Applicants have identified andisolated cDNA encoding PRO344 polypeptides, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO344polypeptide have certain homology with the human and mouse complementproteins. Accordingly, it is presently believed that the PRO344polypeptide disclosed in the present application is a newly identifiedmember of the complement family and possesses the ability to affect theinflammation process as is typical of the complement family of proteins.

[0259] 8. Full-length PRO347 Polypeptides

[0260] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO347. In particular, Applicants have identified andisolated cDNA encoding a PRO347 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that portions of the PRO347polypeptide have certain homology with various cysteine-rich secretoryproteins. Accordingly, it is presently believed that PRO347 polypeptidedisclosed in the present application is a newly identified cysteine-richsecretory protein and may possess activity typical of the cysteine-richsecretory protein family.

[0261] 9. Full-length PRO354 Polypeptides

[0262] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO354. In particular, Applicants have identified andisolated cDNA encoding a PRO354 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that portions of the PRO354polypeptide have certain homology with the inter-alpha-trypsin inhibitorheavy chain protein. Accordingly, it is presently believed that PRO354polypeptide disclosed in the present application is a newly identifiedinter-alpha-trypsin inhibitor heavy chain homolog.

[0263] 10. Full-length PRO355 Polypeptides

[0264] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO355. In particular, Applicants have identified andisolated cDNA encoding a PRO355 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs. Applicants found that various portions of the PRO355polypeptide have certain homology with the CRTAM protein. Applicantshave also found that the DNA encoding the PRO355 polypeptide also hashomology to the thymocyte activation and developmental protein, the H20Areceptor, the H20B receptor, the poliovirus receptor and theCercopithecus aethiops AGM delta 1 protein. Accordingly, it is presentlybelieved that PRO355 polypeptide disclosed in the present application isa newly identified member of the CRTAM protein family.

[0265] 11. Full-length PRO357 Polypeptides

[0266] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO357. In particular, Applicants have identified andisolated cDNA encoding a PRO357 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO357polypeptide have certain homology with the acid labile subunit ofinsulin-like growth factor. Applicants have also found that non-codingregions of the DNA44804-1248 align with a human gene signature asdescribed in WO 95114772. Applicants have further found that non-codingregions of the DNA44804-1248 align with the adenovirus type 12/humanrecombinant viral DNA as described in Deuring and Doerfler, Gene,26:283-289 (1983). Based on the coding region homology, it is presentlybelieved that PRO357 polypeptide disclosed in the present application isa newly identified member of the leucine rich repeat family of proteins,and particularly, is related to the acid labile subunit of insulin-likegrowth factor. As such, PRO357 is likely to be involved in bindingmechanisms, and may be part of a complex.

[0267] 12. Full-length PRO715 Polypeptides

[0268] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO715. In particular, Applicants have identified andisolated cDNA molecules encoding PRO715 polypeptides, as disclosed infurther detail in the Examples below. Using BLAST and FastA sequencealignment computer programs, Applicants found that various portions ofthe PRO715 polypeptides have certain homology with the various membersof the tumor necrosis family of proteins. Accordingly, it is presentlybelieved that the PRO715 polypeptides disclosed in the presentapplication are newly identified members of the tumor necrosis factorfamily of proteins.

[0269] 13. Full-length PRO353 Polypeptides

[0270] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO353. In particular, Applicants have identified andisolated cDNA encoding PRO353 polypeptides, as disclosed in furtherdetail in the Examples below. Using BLAST and, FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO353polypeptides have certain homology with the human and mouse complementproteins. Accordingly, it is presently believed that the PRO353polypeptides disclosed in the present application are newly identifiedmembers of the complement protein family and possesses the ability toeffect the inflammation process as is typical of the complement familyof proteins.

[0271] 14. Full-length PRO361 Polypeptides

[0272] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO361. In particular, Applicants have identified andisolated cDNA encoding a PRO361 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO361polypeptide have certain homology with the mucin and chitinase proteins.Accordingly, it is presently believed that PRO361 polypeptide disclosedin the present application is a newly identified member of the mucinand/or chitinase protein families and may be associated with cancer,plant pathogenesis or receptor functions typical of the mucin andchitinase protein families, respectively.

[0273] 15. Full-length PRO365 Polypeptides

[0274] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO365. In particular, Applicants have identified andisolated cDNA encoding a PRO365 polypeptide, as disclosed in furtherdetail in the Examples below. Using BLAST and FastA sequence alignmentcomputer programs, Applicants found that various portions of the PRO365polypeptide have certain homology with the human 2-19 protein.Accordingly, it is presently believed that PRO365 polypeptide disclosedin the present application is a newly identified member of the human2-19 protein family.

[0275] 2. PRO Polypeptide Variants

[0276] In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

[0277] Variations in the native full-length sequence PRO or in variousdomains of the PRO described herein, can be made, for example, using anyof the techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

[0278] PRO polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

[0279] PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

[0280] In particular embodiments, conservative substitutions of interestare shown in Table 1 under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leunorleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrleu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;norleucine

[0281] Substantial modifications in function or immunological identityof the PRO polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0282] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0283] (2) neutral hydrophilic: cys, ser, thr;

[0284] (3) acidic: asp, glu;

[0285] (4) basic: asn, gln, his, lys, arg;

[0286] (5) residues that influence chain orientation: gly, pro; and

[0287] (6) aromatic: trp, tyr, phe.

[0288] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0289] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res. 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

[0290] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol. 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

[0291] C. Modifications of PRO

[0292] Covalent modifications of PRO are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a PRO polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C- terminal residues of the PRO. Derivatization withbifunctional agents is useful, for instance, for cross inking PRO to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0293] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0294] Another type of covalent modification of the PRO polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

[0295] Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

[0296] Another means of increasing the number of carbohydrate moietieson the PRO polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0297] Removal of carbohydrate moieties present on the PRO polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.138:350 (1987).

[0298] Another type of covalent modification of PRO comprises linkingthe PRO polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0299] The PRO of the present invention may also be modified in a way toform a chimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

[0300] In one embodiment, such a chimeric molecule comprises a fusion ofthe PRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl- terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology. 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0301] In an alternative embodiment, the chimeric molecule may comprisea fusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0302] D. Preparation of PRO

[0303] The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Pepitide Synthesis W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

[0304] 1. Isolation of DNA Encoding PRO

[0305] DNA encoding PRO may be obtained from a cDNA library preparedfrom tissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

[0306] Libraries can be screened with probes (such as antibodies to thePRO or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

[0307] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0308] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined using methods known in the art and as described herein.

[0309] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0310] 2. Selection and Transformation of Host Cells

[0311] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

[0312] Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published June 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyomithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature. 336:348-352 (1988).

[0313] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published April 12, 1989), Pseudomonas such asP. aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3phoA E15 (argF-lac)169degP ompT kan^(r) ; E. coli W3110 strain 37D6, which has the completegenotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan^(r) ;E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycinresistant degP deletion mutation; and an E. coli strain having mutantperiplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued Aug. 7,1990. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

[0314] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forPRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host microorganism. Others include Schizosaccharomyces pombe(Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published May 2,1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K-bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

[0315] Suitable host cells for the expression of glycosylated PRO arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

[0316] 3. Selection and Use of a Replicable Vector

[0317] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

[0318] The PRO may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

[0319] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0320] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0321] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics 85:12 (1977)].

[0322] Expression and cloning vectors usually contain a promoteroperably linked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

[0323] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase,pyruvate decarboxylase,phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphateisomerase, phosphoglucose isomerase, andglucokinase.

[0324] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0325] PRO transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40), from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, and fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

[0326] Transcription of a DNA encoding the PRO by higher eukaryotes maybe increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

[0327] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding PRO.

[0328] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of PRO in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:4046 (1979); EP 117,060; and EP 117,058.

[0329] 4. Detecting Gene Amplification/Expression

[0330] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0331] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

[0332] 5. Purification of Polypeptide

[0333] Forms of PRO may be recovered from culture medium or from hostcell lysates. If membrane-bound, it can be released from the membraneusing a suitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

[0334] It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice. Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

[0335] E. Uses for PRO

[0336] Nucleotide sequences (or their complement) encoding PRO havevarious applications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

[0337] The full-length native sequence PRO gene, or portions thereof,may be used as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

[0338] Any EST sequences disclosed in the present application maysimilarly be employed as probes, using the methods disclosed herein.

[0339] Other useful fragments of the PRO nucleic acids include antisenseor sense oligonucleotides comprising a singe-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target PRO mRNA(sense) or PRO DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of PRO DNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 to 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0340] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranscription or translation of the target sequence by one of severalmeans, including enhanced degradation of the duplexes, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression of PROproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0341] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10048, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0342] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

[0343] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0344] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0345] Antisense or sense RNA or DNA molecules are generally at leastabout 5 bases in length, about 10 bases in length, about 15 bases inlength, about 20 bases in length, about 25 bases in length, about 30bases in length, about 35 bases in length, about 40 bases in length,about 45 bases in length, about 50 bases in length, about 55 bases inlength, about 60 bases in length, about 65 bases in length, about 70bases in length, about 75 bases in length, about 80 bases in length,about 85 bases in length, about 90 bases in length, about 95 bases inlength, about 100 bases in length, or more.

[0346] The probes may also be employed in PCR techniques to generate apool of sequences for identification of closely related PRO codingsequences.

[0347] Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

[0348] When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

[0349] Nucleic acids which encode PRO or its modified forms can also beused to generate either transgenic animals or “knock out” animals which,in turn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

[0350] Alternatively, non-human homologues of PRO can be used toconstruct a PRO “knock out” animal which has a defective or altered geneencoding PRO as a result of homologous recombination between theendogenous gene encoding PRO and altered genomic DNA encoding PROintroduced into an embryonic stem cell of the animal. For example, cDNAencoding PRO can be used to clone genomic DNA encoding PRO in accordancewith established techniques. A portion of the genomic DNA encoding PROcan be deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the PRO polypeptide.

[0351] Nucleic acid encoding the PRO polypeptides may also be used ingene therapy. In gene therapy applications, genes are introduced intocells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

[0352] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology 11, 205-210 [1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0353] The PRO polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes and theisolated nucleic acid sequences may be used for recombinantly expressingthose markers.

[0354] The nucleic acid molecules encoding the PRO polypeptides orfragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identifynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachPRO nucleic acid molecule of the present invention can be used as achromosome marker.

[0355] The PRO polypeptides and nucleic acid molecules of the presentinvention may also be used for tissue typing, wherein the PROpolypeptides of the present invention may be differentially expressed inone tissue as compared to another. PRO nucleic acid molecules will finduse for generating probes for PCR, Northern analysis, Southern analysisand Western analysis.

[0356] The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulin; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

[0357] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

[0358] Therapeutic compositions herein generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0359] The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

[0360] Dosages and desired drug concentrations of pharmaceuticalcompositions of the present invention may vary depending on theparticular use envisioned. The determination of the appropriate dosageor route of administration is well within the skill of an ordinaryphysician. Animal experiments provide reliable guidance for thedetermination of effective doses for human therapy. Interspecies scalingof effective doses can be performed following the principles laid downby Mordenti, J. and Chappell, W. “The use of interspecies scaling intoxicokinetics” In Toxicokinetics and New Drug Development, Yacobi etal., Eds., Pergamon Press, New York 1989, pp. 42-96.

[0361] When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

[0362] Where sustained-release administration of a PRO polypeptide isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon- (rhIFN- ), interleukin-2, and MN rgp120. Johnson et al.,Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993);Hora et al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

[0363] The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 141.

[0364] This invention encompasses methods of screening compounds toidentify those that mimic the PRO polypeptide (agonists) or prevent theeffect of the PRO polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the PRO polypeptides encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

[0365] The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

[0366] All assays for antagonists are common in that they call forcontacting the drug candidate with a PRO polypeptide encoded by anucleic acid identified herein under conditions and for a timesufficient to allow these two components to interact.

[0367] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the PRO polypeptide encoded by the geneidentified herein or the drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.Non-covalent attachment generally is accomplished by coating the solidsurface with a solution of the PRO polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the PRO polypeptide to be immobilized can be used to anchorit to a solid surface. The assay is performed by adding thenon-immobilized component, which may be labeled by a detectable label,to the immobilized component, e.g., the coated surface containing theanchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

[0368] If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London). 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

[0369] Compounds that interfere with the interaction of a gene encodinga PRO polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows. usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

[0370] To assay for antagonists, the PRO polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

[0371] As an alternative approach for receptor identification, labeledPRO polypeptide can be photoaffinity-linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE and exposed to X-ray film. The labeledcomplex containing the receptor can be excised, resolved into peptidefragments, and subjected to protein micro-sequencing. The amino acidsequence obtained from micro- sequencing would be used to design a setof degenerate oligonucleotide probes to screen a cDNA library toidentify the gene encoding the putative receptor.

[0372] In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

[0373] More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

[0374] Another potential PRO polypeptide antagonist is an antisense RNAor DNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al.,Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

[0375] Potential antagonists include small molecules that bind to theactive site, the receptor binding site, or growth factor or otherrelevant binding site of the PRO polypeptide, thereby blocking thenormal biological activity of the PRO polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

[0376] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g., Rossi, Current Biology. 4:469471 (1994), and PCTpublication No. WO 97/33551 (published Sep. 18, 1997).

[0377] Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

[0378] These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0379] PRO241 polypeptides of the present invention which possessbiological activity related to that of the endogenous biglycan proteinmay be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO241 polypeptides of the present invention for such purposes.

[0380] Chordin is a candidate gene for a dysmorphia syndrome known asCornelia de Lange Syndrome (CDL) which is characterized by distinctivefacial features (low anterior hairline, synophrys, antenerted nares,maxillary prognathism, long philtrum, ‘carp’ mouth), prenatal andpostnatal growth retardation, mental retardation and, often but notalways, upper limb abnormalities. There are also rare cases where CDL ispresent in association with thrombocytopenia. The gene for CDL has beenmapped by linkage to 3q26.3 (OMIM #122470). Xchd involvement in earlyXenopus patterning and nervous system development makes CHD inintriguing candidate gene. CHD maps to the appropriate region onchromosome 3. It is very close to THPO, and deletions encompassing bothTHPO and CHD could result in rare cases of thrombocytopenia anddevelopmental abnormalities. In situ analysis of CD revealed that almostall adult tissues are negative for CHD expression, the only positivesignal was observed in the cleavage line of the developing synovialjoint forming between the femoral head and acetabulum (hip joint)implicating CHD in the development and presumably growth of long bones.Such a function, if disrupted, could result in growth retardation.

[0381] The human CHD amino acid sequence predicted from the cDNA is 50%identical (and 66% conserved) to Xchd. All 40 cysteines in the 4cysteine-rich domains are conserved. These cysteine rich domains aresimilar to those observed in thrombospondin, procollagen and vonWillebrand factor. Bornstein, P. FASEB J 6: 3290-3299 (1992); Hunt, L. &Barker, W. Biochem. Biophys. Res. Commun. 144: 876-882 (1987).

[0382] The human CHD locus (genomic PRO243) comprises 23 exons in 9.6 kbof genomic DNA. The initiating methionine is in exon 1 and the stopcodon in exon 23. A CpG island is located at the 5′ and of the gene,beginning approximately 100 bp 5′ of exon 1 and extends through thefirst exon and ends within the first intron. The THPO and CHD loci areorganized in a head-to-head fashion with approximately 2.2 kb separatingtheir transcription start sites. At the protein level, PRO243 is 51%identical to Xenopus chordin (Xchd). All forty cysteines in the oneamino terminal and three carboxy terminal cysteine-rich clusters areconserved.

[0383] PRO243 is a 954 amino acid polypeptide having a signal sequenceat residues 1 to about 23. There are 4 cysteine clusters: (1) residuesabout 51 to about 125; (2) residues about 705 to about 761; (3) residuesabout 784 to about 849; and (4) residues about 897 to about 931. Thereare potential leucine zippers at residues about 315 to about 396, andN-glycosylation sites at residues 217, 351, 365 and 434.

[0384] PRO299 polypeptides and portions thereof which have homology tothe notch protein may be useful for in vivo therapeutic purposes, aswell as for various other applications. The identification of novelnotch proteins and related molecules may be relevant to a number ofhuman disorders such as those effecting development. Thus, theidentification of new notch proteins and notch-like molecules is ofspecial importance in that such proteins may serve as potentialtherapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO299.

[0385] PRO323 polypeptides of the present invention which possessbiological activity related to that of one or more endogenousdipeptidase proteins may be employed both in vivo for therapeuticpurposes and in vitro. Those of ordinary skill in the art will well knowhow to employ the PRO323 polypeptides of the present invention for suchpurposes.

[0386] PRO327 polypeptides of the present invention which possessbiological activity related to that of the endogenous prolactin receptorprotein may be employed both in vivo for therapeutic purposes and invitro. Those of ordinary skill in the art will well know how to employthe PRO327 polypeptides of the present invention for such purposes.PRO327 polypeptides which possess the ability to bind to prolactin mayfunction both in vitro and in vivo as prolactin antagonists.

[0387] PRO233 polypeptides and portions thereof which have homology toreductase may also be useful for in vivo therapeutic purposes, as wellas for various other applications. The identification of novel reductaseproteins and related molecules may be relevant to a number of humandisorders such as inflammatory disease, organ failure, atherosclerosis,cardiac injury, infertility, birth defects, premature aging, AIDS,cancer, diabetic complications and mutations in general. Given thatoxygen free radicals and antioxidants appear to play important roles ina number of disease processes, the identification of new reductaseproteins and reductase-like molecules is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research, as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO233.

[0388] PRO344 polypeptides and portions thereof which have homology tocomplement proteins may also be useful for in vivo therapeutic purposes,as well as for various other applications. The identification of novelcomplement proteins and related molecules may be relevant to a number ofhuman disorders such as effecting the inflammatory response of cells ofthe immune system. Thus, the identification of new complement proteinsand complement-like molecules is of special importance in that suchproteins may serve as potential therapeutics for a variety of differenthuman disorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO344.

[0389] PRO347 polypeptides of the present invention which possessbiological activity related to that of cysteine-rich secretory proteinsmay be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO347 polypeptides of the present invention for such purposes.

[0390] PRO354 polypeptides of the present invention which possessbiological activity related to that of the heavy chain of theinter-alpha-trypsin inhibitor protein may be employed both in vivo fortherapeutic purposes and in vitro. Those of ordinary skill in the artwill well know how to employ the PRO354 polypeptides of the presentinvention for such purposes.

[0391] PRO355 polypeptides and portions thereof which have homology toCRTAM may also be useful for in vivo therapeutic purposes, as well asfor various other applications. The identification of novel moleculesassociated with T cells may be relevant to a number of human disorderssuch as conditions involving the immune system in general. Given thatthe CRTAM protein binds antibodies which play important roles in anumber of disease processes, the identification of new CRTAM proteinsand CRTAM-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research, as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO355.

[0392] PRO357 can be used in competitive binding assays with ALS todetermine its activity with respect to ALS. Moreover, PRO357 can be usedin assays to determine if it prolongs polypeptides which it may complexwith to have longer half-lives in vivo. PRO357 can be used similarly inassays with carboxypeptidase, to which it also has homology. The resultscan be applied accordingly.

[0393] PRO715 polypeptides of the present invention which possessbiological activity related to that of the tumor necrosis factor familyof proteins may be employed both in vivo for therapeutic purposes and invitro. Those of ordinary skill in the art will well know how to employthe PRO715 polypeptides of the present invention for such purposes.PRO715 polypeptides will be expected to bind to their specificreceptors, thereby activating such receptors. Variants of the PRO715polypeptides of the present invention may function as agonists orantagonists of their specific receptor activity.

[0394] PRO353 polypeptides and portions thereof which have homology tothe complement protein may also be useful for in vivo therapeuticpurposes, as well as for various other applications. The identificationof novel complement proteins and related molecules may be relevant to anumber of human disorders such as effecting the inflammatory response ofcells of the immune system. Thus, the identification of new complementproteins complement-like molecules is of special importance in that suchproteins may serve as potential therapeutics for a variety of differenthuman disorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO353.

[0395] PRO361 polypeptides and portions thereof which have homology tomucin and/or chitinase proteins may also be useful for in vivotherapeutic purposes, as well as for various other applications. Theidentification of novel mucin and/or chitinase proteins and relatedmolecules may be relevant to a number of human disorders such as canceror those involving cell surface molecules or receptors. Thus, theidentification of new mucin and/or chitinase proteins is of specialimportance in that such proteins may serve as potential therapeutics fora variety of different human disorders. Such polypeptides may also playimportant roles in biotechnological and medical research as well asvarious industrial applications. As a result, there is particularscientific and medical interest in new molecules, such as PRO361.

[0396] PRO365 polypeptides and portions thereof which have homology tothe human 2-19 protein may also be useful for in vivo therapeuticpurposes, as well as for various other applications. The identificationof novel human 2-19 proteins and related molecules may be relevant to anumber of human disorders such as modulating the binding or activity ofcells of the immune system. Thus, the identification of new human 2-19proteins and human 2-19 protein-like molecules is of special importancein that such proteins may serve as potential therapeutics for a varietyof different human disorders. Such polypeptides may also play importantroles in biotechnological and medical research as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO365.

[0397] F. Anti-PRO Antibodies

[0398] The present invention further provides anti-PRO antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

[0399] 1. Polyclonal Antibodies

[0400] The anti-PRO antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0401] 2. Monoclonal Antibodies

[0402] The anti-PRO antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0403] The immunizing agent will typically include the PRO polypeptideor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0404] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeuretal., Monoclonal Antibody Production Techniques and Applications. MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0405] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst PRO. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem. 107:220 (1980).

[0406] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0407] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0408] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0409] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0410] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0411] 3. Human and Humanized Antibodies

[0412] The anti-PRO antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann etal., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)].

[0413] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import”variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al. Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0414] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0415] 4. Bispecific Antibodies

[0416] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the PRO, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

[0417] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature 305:537-539 (1983)]. Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published May13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0418] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology. 121:210 (1986).

[0419] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0420] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0421] Fab′ fragments may be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

[0422] Various technique for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

[0423] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

[0424] Exemplary bispecific antibodies may bind to two differentepitopes on a given PRO polypeptide herein. Alternatively, an anti-PROpolypeptide arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular PRO polypeptide.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a particular PRO polypeptide. These antibodiespossess a PRO-binding arm and an arm which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the PRO polypeptide and furtherbinds tissue factor (TF).

[0425] 5. Heteroconjugate Antibodies

[0426] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0427] 6. Effector Function Engineering

[0428] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

[0429] 7. Immunoconjugates

[0430] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0431] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹IN, ⁹⁰Y,and ¹⁸⁶Re.

[0432] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0433] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0434] 8. Immunoliposomes

[0435] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0436] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, andPEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extrudedthrough filters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989).

[0437] 9. Pharmaceutical Compositions of Antibodies

[0438] Antibodies specifically binding a PRO polypeptide identifiedherein, as well as other molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders in the form of pharmaceutical compositions.

[0439] If the PRO polypeptide is intracellular and whole antibodies areused as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The herein may also contain more thanone active compound as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0440] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0441] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0442] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecularS-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0443] G. Uses for anti-PRO Antibodies

[0444] The anti-PRO antibodies of the invention have various utilities.For example, anti-PRO antibodies may be used in diagnostic assays forPRO, e.g., detecting its expression in specific cells, tissues, orserum. Various diagnostic assay techniques known in the art may be used,such as competitive binding assays, direct or indirect sandwich assaysand immunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques.CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0445] Anti-PRO antibodies also are useful for the affinity purificationof PRO from recombinant cell culture or natural sources. In thisprocess, the antibodies against PRO are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art. The immobilized antibody then is contacted with a samplecontaining the PRO to be purified, and thereafter the support is washedwith a suitable solvent that will remove substantially all the materialin the sample except the PRO, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solventthat will release the PRO from the antibody.

[0446] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0447] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0448] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, Va.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

[0449] The extracellular domain (ECD) sequences (including the secretionsignal sequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

[0450] Using this extracellular domain homology screen, consensus DNAsequences were assembled relative to the other identified EST sequencesusing phrap. In addition, the consensus DNA sequences obtained wereoften (but not always) extended using repeated cycles of BLAST orBLAST-2 and phrap to extend the consensus sequence as far as possibleusing the sources of EST sequences discussed above.

[0451] Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

[0452] The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

Example 2 Isolation of cDNA clones by Amylase Screening

[0453] 1. Preparation of Oligo dT Primed cDNA Library

[0454] mRNA was isolated from a human tissue of interest using reagentsand protocols from Invitrogen, San Diego, Calif. (Fast Track 2). ThisRNA was used to generate an oligo dT primed cDNA library in the vectorpRK5D using reagents and protocols from Life Technologies, Gaithersburg,Md. (Super Script Plasmid System). In this procedure, the doublestranded cDNA was sized to greater than 1000 bp and the SalI/NotITinkered cDNA was cloned into XhoI/NotI cleaved vector. pRK5D is acloning vector that has an sp6 transcription initiation site followed byan SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloningsites.

[0455] 2. Preparation of Random Primed cDNA Library

[0456] A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

[0457] 3. Transformation and Detection

[0458] DNA from the library described in paragraph 2 above was chilledon ice to which was added electrocompetent DH10B bacteria (LifeTechnologies, 20 ml). The bacteria and vector mixture was thenelectroporated as recommended by the manufacturer. Subsequently, SOCmedia (Life Technologies, 1 ml) was added and the mixture was incubatedat 37° C. for 30 minutes. The transformants were then plated onto 20standard 150 mm LB plates containing ampicillin and incubated for 16hours (37° C.). Positive colonies were scraped off the plates and theDNA was isolated from the bacterial pellet using standard protocols,e.g. CsCl-gradient. The purified DNA was then carried on to the yeastprotocols below.

[0459] The yeast methods were divided into three categories: (1)Transformation of yeast with the plasmid/cDNA combined vector; (2)Detection and isolation of yeast clones secreting amylase; and (3) PCRamplification of the insert directly from the yeast colony andpurification of the DNA for sequencing and further analysis.

[0460] The yeast strain used was HD56-5A (ATCC-90785). This strain hasthe following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

[0461] Transformation was performed based on the protocol outlined byGietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed cells werethen inoculated from agar into YEPD complex media broth (100 ml) andgrown overnight at 30° C. The YEPD broth was prepared as described inKaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., p. 207 (1994). The overnight culture was thendiluted to about 2×10⁶ cells/ml (approx. OD₆₀₀ =O.1) into fresh YEPDbroth (500 ml) and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀ =O.4-0.5).

[0462] The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

[0463] Transformation took place by mixing the prepared cells (100 μl)with freshly denatured single stranded salmon testes DNA (LofstrandLabs, Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl ) inmicrofuge tubes. The mixture was mixed briefly by vortexing, then 40%PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA,100 mM Li₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

[0464] Alternatively, instead of multiple small reactions, thetransformation was performed using a single, large scale reaction,wherein reagent amounts were scaled up accordingly.

[0465] The selective media used was a synthetic complete dextrose agarlacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methodsin Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,p. 208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

[0466] The detection of colonies secreting amylase was performed byincluding red starch in the selective growth media. Starch was coupledto the red dye (Reactive Red-120, Sigma) as per the procedure describedby Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starchwas incorporated into the SCD-Ura agar plates at a final concentrationof 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

[0467] The positive colonies were picked and streaked across freshselective media (onto 150 mm plates) in order to obtain well isolatedand identifiable single colonies. Well isolated single colonies positivefor amylase secretion were detected by direct incorporation of redstarch into buffered SCD-Ura agar. Positive colonies were determined bytheir ability to break down starch resulting in a clear halo around thepositive colony visualized directly.

[0468] 4. Isolation of DNA by PCR Amplification

[0469] When a positive colony was isolated, a portion of it was pickedby a toothpick and diluted into sterile water (30 μl) in a 96 wellplate. At this time, the positive colonies were either frozen and storedfor subsequent analysis or immediately amplified. An aliquot of cells (5μl) was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide I was:

5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′  (SEQ ID NO:16)

[0470] The sequence of reverse oligonucleotide 2 was:

5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′  (SEQ ID NO:17)

[0471] PCR was then performed as follows: a. Denature 92° C., 5 minutesb. 3 cycles of: Denature 92° C., 30 seconds Anneal 59° C., 30 secondsExtend 72° C., 60 seconds c. 3 cycles of: Denature 92° C., 30 secondsAnneal 57° C., 30 seconds Extend 72° C., 60 seconds d. 25 cycles of:Denature 92° C., 30 seconds Anneal 55° C., 30 seconds Extend 72° C., 60seconds e. Hold 4° C.

[0472] The underlined regions of the oligonucleotides annealed to theADH promoter region and the amylase region, respectively, and amplifieda 307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

[0473] Following the PCR, an aliquot of the reaction (5 μl) was examinedby agarose gel electrophoresis in a 1% agarose gel using aTris-Borate-EDTA (TBE) buffering system as described by Sambrook et al.,supra. Clones resulting in a single strong PCR product larger than 400bp were further analyzed by DNA sequencing after purification with a 96Qiaquick PCR clean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Encoding Human PRO241

[0474] A consensus DNA sequence was assembled relative to other ESTsequences as described in Example 1 above. This consensus sequence isherein designated DNA30876. Based on the DNA30876 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO241.

[0475] PCR primers (forward and reverse) were synthesized: forward PCRprimer 5′-GGAAATGAGTGCAAACCCTC-3′ (SEQ ID NO:3) reverse PCR primer5′-TCCCAAGCTGAACACTCATTCTGC-3′ (SEQ ID NO:4)

[0476] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30876 sequence which had the followingnucleotide sequence hybridization probe5′-GGGTGACGGTGTTCCATATCAGAATTGCAGAAGCAAAACTGACCTCAGTT-3′ (SEQ ID NO:5)

[0477] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pair identified above. A positivelibrary was then used to isolate clones encoding the PRO241 gene usingthe probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal kidneytissue (LIB29).

[0478] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO241 [herein designated as DNA34392-1170](SEQ ID NO:1) and the derived protein sequence for PRO241.

[0479] The entire nucleotide sequence of DNA34392-1170 is shown in FIG.1 (SEQ ID NO:1). Clone DNA34392-1170 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 234-236 and ending at the stop codon at nucleotide positions1371-1373 (FIG. 1). The predicted polypeptide precursor is 379 aminoacids long (FIG. 2). The full-length PRO241 protein shown in FIG. 2 hasan estimated molecular weight of about 43,302 daltons and a pI of about7.30. Clone DNA34392-1170 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209526.

[0480] Analysis of the amino acid sequence of the full-length PRO241polypeptide suggests that it possess significant homology to the variousbiglycan proteoglycan proteins, thereby indicating that PRO241 is anovel biglycan homolog polypeptide.

Example 4 Isolation of cDNA Clones Encoding Human PRO243 by GenomicWalking

[0481] Introduction

[0482] Human thrombopoietin (THPO) is a glycosylated hormone of 352amino acids consisting of two domains. The N-terminal domain, sharing50% similarity to erythropoietin, is responsible for the biologicalactivity. The C-terminal region is required for secretion. The gene forthrombopoietin (THPO) maps to human chromosome 3q27-q28 where the sixexons of this gene span 7 kilobase base pairs of genomic DNA (Gurney etal., Blood 85: 981-988 (1995). In order to determine whether there wereany genes encoding THPO homologues located in close proximity to THPO,genomic DNA fragments from this region were identified and sequenced.Three P1 clones and one PAC clones (Genome Systems Inc., St. Louis, Mo.;cat. Nos. P1-2535 and PAC-6539) encompassing the THPO locus wereisolated and a 140 kb region was sequenced using the ordered shotgunstrategy (Chen et al., Genomics 17: 651-656 (1993)), coupled with aPCR-based gap filling approach. Analysis reveals that the region isgene-rich with four additional genes located very close to THPO: tumornecrosis factor-receptor type 1 associated protein 2 (TRAP2) andelongation initiation factor gamma (elF4g), chloride channel 2 (CLCN2)and RNA polymerase II subunit hRPB17. While no THPO homolog was found inthe region, four novel genes have been predicted by computer-assistedgene detection (GRAIL)(Xu et al., Gen. Engin. 16: 241-253 (1994), thepresence of CpG islands (Cross, S. and Bird, A., Curr. Opin. Genet. &Devel. 5: 109-314 (1995), and homology to known genes (as detected byWU-BLAST2.0)(Altschul and Gish, Methods Enzymol. 266: 460-480 (1996)(http://blast.wustl.edu/blast/README.html).

[0483] P1 and PAC Clones

[0484] The initial human P1 clone was isolated from a genomic P1 library(Genome Systems Inc., St. Louis, Mo.; cat. no.: P1-2535) screened withPCR primers designed from the THPO genomic sequence (A. L. Gurney, etal., Blood 85: 981-88 (1995). PCR primers were designed from the endsequences derived from this P1 clone were then used to screen P1 and PAClibraries (Genome Systems, Cat. Nos.: P1-2535 & PAC-6539) to identifyoverlapping clones.

[0485] Ordered Shotgun Strategy

[0486] The Ordered Shotgun Strategy (OSS) (Chen et al., Genomics 17:651-656 (1993)) involves the mapping and sequencing of large genomic DNAclones with a hierarchical approach. The P1 or PAC clone was sonicatedand the fragments subcloned into lambda vector (λBluestar) (Novagen,Inc., Madison, Wis.; cat. no. 69242-3). The lambda subclone inserts wereisolated by long-range PCR (Barnes, W. Proc. Natl. Acad. Sci. USA 91:2216-2220 (1994) and the ends sequenced. The lambda-end sequences wereoverlapped to create a partial map of the original clone. Those lambdaclones with overlapping end-sequences were identified, the insetssubcloned into a plasmid vector (pUC9 or pUC 18) and the ends of theplasmid subclones were sequenced and assembled to generate a contiguoussequence. This directed sequencing strategy minimizes the redundancyrequired while allowing one to scan for and concentrate on interestingregions.

[0487] In order to define better the THPO locus and to search for othergenes related to the hematopoietin family, four genomic clones wereisolated from this region by PCR screening of human P1 and PAC libraries(Genome System, Inc., Cat. Nos.: P1-2535 and PAC-6539). The sizes of thegenomic fragments are as follows: P1.t is 40 kb; P1.g is 70 kb; P1.u is70 kb; and PAC.z is 200 kb. Approximately 80% of the 200 kb genomic DNAregion was sequenced by the Ordered Shotgun Strategy (OSS) (Chen et al.,Genomics 17: 651-56 (1993), and assembled into contigs usingAutoAssembler™ (Applied Biosystems, Perkin Elmer, Foster City, Calif.,cat. no. 903227). The preliminary order of these contigs was determinedby manual analysis. There were 46 contigs and filling in the gaps wasemployed. Table 7 summarized the number and sizes of the gaps. TABLE 7Summary of the gaps in the 140 kb region Size of gap number <50 bp 13 50-150 bp  7 150-300 bp  7  300-1000 bp 10 1000-5000 bp  7 >5000 bp  2(15,000 bp)

[0488] DNA Sequencing

[0489] ABI DYE-primer™ chemistry (PE Applied Biosystems, Foster City,Calif.; Cat. No.: 402112) was used to end-sequence the lambda andplasmid subclones. ABI DYE-terminater™ chemistry (PE Applied Biosystems,Foster City, Calif., Cat. No: 403044) was used to sequence the PCRproducts with their respective PCR primers. The sequences were collectedwith an ABI377 instrument. For PCR products larger than 1 kb, walkingprimers were used. The sequences of contigs generated by the OSSstrategy in AutoAssembler™ a (PE Applied Biosystems, Foster City,Calif.; Cat. No: 903227) and the gap-filling sequencing trace files wereimported into Sequencher™ (Gene Codes Corp., Ann Arbor, Mich.) foroverlapping and editing.

[0490] PCR-Based Gap Filling Strategy

[0491] Primers were designed based on the 5′- and 3′-end sequenced ofeach contig, avoiding repetitive and low quality sequence regions. Allprimers were designed to be 19-24-mers with 50-70% G/C content. Oligoswere synthesized and gel-purified by standard methods.

[0492] Since the orientation and order of the contigs were unknown,permutations of the primers were used in the amplification reactions.Two PCR kits were used: first, XL PCR kit (Perkin Elmer, Norwalk, Conn.;Cat. No.: N8080205), with extension times of approximately 10 minutes;and second, the Taq polymerase PCR kit (Qiagen Inc., Valencia, Calif.;Cat. No.: 201223) was used under high stringency conditions if smearedor multiple products were observed with the XL PCR kit. The main PCRproduct from each successful reactions was extracted from a 0.9% lowmelting agarose gel and purified with the Geneclean DNA Purification kitprior to sequencing.

[0493] Analysis

[0494] The identification and characterization of coding regions wascarried out as follows: First, repetitive sequences were masked usingRepeatMasker (A. F. A. Smit & P. Green,http://ftp.genome.washington.edu/RM/RM_details.html)which screens DNAsequences in FastA format against a library of repetitive elements andreturns a masked query sequence. Repeats not masked were identified bycomparing the sequence to the GenBank database using WUBLAST (Altschul,S & Gish, W., Methods Enzymol. 266: 460-480 (1996) and were maskedmanually.

[0495] Next, known genes were revealed by comparing the genomic regionsagainst Genentech's protein database using the WUBLAST2.0 algorithm andthen annotated by aligning the genomic and cDNA sequences for each gene,respectively, using a Needleman-Wunch (Needleman and Wunsch, J. Mol.Biol. 48: 443-453 (1970) algorithm to find regions of local identitybetween sequences which are otherwise largely dissimilar. The strategyresults in detection of all exons of the five known genes in the region,THPO, TRAP2, elF4g, CLCN2 and hRPB17 (Table 8). TABLE 8 Summary of knowngenes located in the 140 kb region analyzed Known genes Map positioneukaryotic translation initiation factor 4 gamma 3q27-qterthrombopoietin 3q26-q27 chloride channel 2 3q26-qter TNF receptorassociated protein 2 not previously mapped RNA polymerase II subunithRPB17 not previously mapped

[0496] Finally, novel transcription units were predicted using a numberof approaches. CpG islands (S. Cross & Bird, A., Curr. Opin. Genet. Dev.5: 109-314 (1995) islands were used to define promoter regions and wereidentified as clusters of sites cleaved by enzymes recognizing GC-rich,6 or 8-mer palidromic sequences. CpG islands are usually associated withpromoter regions of genes. WUBLAST2.0 analysis of short genomic regions(10-20 kb) versus GenBank revealed matches to ESTs. The individual ESTsequences (or where possible, their sequence chromatogram files) wereretrieved and assembled with Sequencher to provide a theoretical cDNAsequence (designated herein as DNA34415). GRAIL2 (ApoCom Inc.,Knoxville, Tenn., command line version for the DEC alpha) was used topredict a novel exon. The five known genes in the region served asinternal controls for the success of the GRAIL algorithm.

[0497] Isolation

[0498] Chordin cDNA clones were isolated from an oligo-dT-primed humanfetal lung library. Human fetal lung polyA⁺RNA was purchased fromClontech (cat #6528-1, lot #43777) and 5 mg used to construct a cDNAlibrary in pKR5B (Genentech, LIB26). The 3′-primer(pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT) (SEQ ID NO:8)

[0499] and the 5′-linker (pCGGACGCGTGGGGCCTGCGCACCCAGCT) (SEQ ID NO:9)

[0500] were designed to introduce SalI and NotI restriction sites.Clones were screened with oligonucleotide probes designed from theputative human chordin cDNA sequence (DNA34415) deduced by manually“splicing” together the proposed genomic exons of the gene. PCR primersflanking the probes were used to confirm the identity of the cDNA clonesprior to sequencing.

[0501] The screening oligonucleotides probes were the following: OLI564034415.p1 5′-GCCGCTCCCCGAACGGGCAGCGGCTCCTTCTCAGAA-3′ (SEQ ID NO:10) andOLI5642 34415.p2 5′-GGCGCACAGCACGCAGCGCATCACCCCGAATGGCTC-3′ (SEQ IDNO:11); and

[0502] the flanking probes used were the following: OLI5639 34415.f15′-GTGCTGCCCATCCGTTCTGAGAAGGA-3′ (SEQ ID NO:12) and OLI5643 34415.r5′-GCAGGGTGCTCAAACAGGACAC-3′ (SEQ ID NO:13).

Example 5 Northern Blot and in situ RNA Hybridization Analysis of PRO243

[0503] Expression of PRO243 mRNA in human tissues was examined byNorthern blot analysis. Human polyA+RNA blots derived from human fetaland adult tissues (Clontech, Palo Alto, Calif.; Cat. Nos. 7760-1 and7756-1) were hybridized to a ³²P-labelled cDNA fragments probe based onthe full length PRO243 cDNA. Blots were incubated with the probes inhybridization buffer (5×SSPE; 2×Denhardt's solution; 100 mg/mL denaturedsheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hours at 42° C.The blots were washed several times in 2×SSC; 0.05% SDS for 1 hour atroom temperature, followed by a high stringency wash 30 minute wash in0. 1×SSC; 0.1% SDS at 50° C. and autoradiographed. The blots weredeveloped after overnight exposure by phosphorimager analysis (Fuji).

[0504] PRO243 mRNA transcripts were detected. Analysis of the expressionpattern showed the strongest signal of the expected 4.0 kb transcript inadult and fetal liver and a very faint signal in the adult kidney. Fetalbrain, lung and kidney were negative, as were adult heart, brain, lungand pancreas. Smaller transcripts were observed in placenta (2.0 kb),adult skeletal muscle (1.8 kb) and fetal liver (2.0 kb).

[0505] In situ hybridization of adult human tissue of PRO243 gave apositive signal in the cleavage line of the developing synovial jointforming between the femoral head and acetabulum. All other tissues werenegative. Additional sections of human fetal face, head, limbs and mouseembryos were examined. Expression in human fetal tissues was observedadjacent to developing limb and facial bones in the perosteal msenchyme.The expression was highly specific and was often adjacent to areasundergoing vascularization. Expression was also observed in thedeveloping temporal and occipital lobes of the fetal brain, but was notobserved elsewhere in the brain. In addition, expression was seen in theganglia of the developing inner ear. No expression was seen in any ofthe mouse tissues with the human probes.

[0506] In situ hybridization was performed using an optimized protocol,using PCR-generating ³³P-labeled riboprobes. (Lu and Gillett, CellVision 1: 169-176 (1994)). Formalin-fixed, paraffin-embedded human fetaland adult tissues were sectioned, deparaffinized, deproteinated inproteinase K (20 g/ml) for 15 minutes at 37° C., and further processedfor in situ hybridization as described by Lu and Gillett (1994). A[³³P]-UTP-labeled antisense riboprobe was generated from a PCR productand hybridized at 55° C. overnight. The slides were dipped in Kodak NTB2nuclear track emulsion and exposed for 4 weeks.

Example 6 Isolation of cDNA clones Encoding Human PRO299

[0507] A cDNA sequence designated herein as DNA28847 (FIG. 7; SEQ IDNO:18) was isolated as described in Example 2 above. After furtheranalysis, a 3′ truncated version of DNA28847 was found and is hereindesignated DNA35877 (FIG. 8; SEQ ID NO:19). Based on the DNA35877sequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO299.Forward and reverse PCR primers generally range from 20 to 30nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

[0508] Forward and reverse PCR primers were synthesized: forward PCRprimer 5′-CTCTGGAAGGTCACGGCCACAGG-3′ (SEQ ID NO:20) reverse PCR primer5′-CTCAGTTCGGTTGGCAAAGCTCTC-3′ (SEQ ID NO:21)

[0509] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA35877 sequence which had the followingnucleotide sequence hybridization probe5′-CAGTGCTCCCTCATAGATGGACGAAAGTGTGACCCCCCTTTCAGGCGAGAGCTTTGCCAACCG (SEQID NO:22) AACTGA-3′

[0510] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with one of the PCR primer pairs identified above. Apositive library was then used to isolate clones encoding the PRO299sequence using the probe oligonucleotide.

[0511] RNA for construction of the cDNA libraries was isolated fromhuman fetal brain tissue. The cDNA libraries used to isolate the cDNAclones were constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

[0512] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO299 [herein designated as DNA39976-1215](SEQ ID NO:14) and the derived protein sequence for PRO299.

[0513] The entire nucleotide sequence of DNA39976-1215 is shown in FIG.5 (SEQ ID NO:14). Clone DNA39976-1215 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 111-113 and ending at the stop codon at nucleotide positions2322-2324 (FIG. 5). The predicted polypeptide precursor is 737 aminoacids long (FIG. 6). Important regions of the polypeptide sequenceencoded by clone DNA39976-1215 have been identified and include thefollowing: a signal peptide corresponding to amino acids 1-28, aputative transmembrane region corresponding to amino acids 638-662, 10EGF repeats, corresponding to amino acids 80-106, 121-203, 336-360,378-415, 416-441, 454-490, 491-528, 529-548, 567-604, and 605-622,respectively, and 10 potential N-glycosylation sites, corresponding toamino acids 107-120, 204-207, 208-222, 223-285, 286-304, 361-374,375-377, 442-453, 549-563, and 564-566, respectively. CloneDNA39976-1215 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209524.

[0514] Analysis of the amino acid sequence of the full-length PRO299polypeptide suggests that portions of it possess significant homology tothe notch protein, thereby indicating that PRO299 may be a novel notchprotein homolog and have activity typical of the notch protein.

Example 7 Isolation of cDNA Clones Encoding Human PRO323

[0515] A consensus DNA sequence was assembled relative to other ESTsequences as described in Example 1 above. This consensus sequence isherein designated DNA30875. Based on the DNA30875 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO323.

[0516] PCR primers (two forward and one reverse) were synthesized:forward PCR primer 1 5′-AGTTCTGGTCAGCCTATGTGCC-3′ (SEQ ID NO:25) forwardPCR primer 2 5′-CGTGATGGTGTCTTTGTCCATGGG-3′ (SEQ ID NO:26) reverse PCRprimer 5′-CTCCACCAATCCCGATGAACTTGG-3′ (SEQ ID NO:27)

[0517] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30875 sequence which had the followingnucleotide sequence

[0518] hybridization probe

5′-GAGCAGATTGACCTCATACGCCGCATGTGTGCCTCCTATTCTGAGCTGGA-3′  (SEQ ID NO:28)

[0519] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pairs identified above. A positivelibrary was then used to isolate clones encoding the PRO323 gene usingthe probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal livertissue (LIB6).

[0520] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO323 [herein designated as DNA35595-1228](SEQ ID NO:23) and the derived protein sequence for PRO323.

[0521] The entire nucleotide sequence of DNA35595-1228 is shown in FIG.9 (SEQ ID NO:23). Clone DNA35595-1228 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 110-112 and ending at the stop codon at nucleotide positions1409-1411 (FIG. 9). The predicted polypeptide precursor is 433 aminoacids long (FIG. 10). The full-length PRO323 protein shown in FIG. 10has an estimated molecular weight of about 47,787 daltons and a pI ofabout 6.11. Clone DNA35595-1228 has been deposited with ATCC and isassigned ATCC deposit no. 209528.

[0522] Analysis of the amino acid sequence of the full-length PRO323polypeptide suggests that portions of it possess significant homology tovarious dipeptidase proteins, thereby indicating that PRO323 may be anovel dipeptidase protein.

Example 8 Isolation of cDNA Clones Encoding Human PRO327

[0523] An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and various ESTsequences were identified which showed certain degrees of homology tohuman prolactin receptor protein. Those EST sequences were aligned usingphrap and a consensus sequence was obtained. This consensus DNA sequencewas then extended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using the sources of EST sequencesdiscussed above. The extended assembly sequence is herein designatedDNA38110. The above searches were performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzmolog 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

[0524] Based upon the DNA38110 consensus sequence obtained as describedabove, oligonucleotides were synthesized: 1) to identify by PCR a cDNAlibrary that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO327.

[0525] PCR primers (forward and reverse) were synthesized as follows:forward PCR primer 5′-CCCGCCCGACGTGCACGTGAGCC-3′ (SEQ ID NO:33) reversePCR primer 5′-TGAGCCAGCCCAGGAACTGCTTG-3′ (SEQ ID NO:34)

[0526] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA381 10 consensus sequence which hadthe following nucleotide sequence hybridization probe5′-CAAGTGCGCTGCAACCCCTTTGGCATCTATGGCTCCAAGAAAGCCGGGAT-3′ (SEQ ID NO:35)

[0527] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pair identified above. A positivelibrary was then used to isolate clones encoding the PRO327 gene usingthe probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal lungtissue (LIB26).

[0528] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO327 [herein designated as DNA38113-1230](SEQ ID NO:16) and the derived protein sequence for PRO327.

[0529] The entire nucleotide sequence of DNA38113-1230 is shown in FIG.13 (SEQ ID NO:31). Clone DNA38113-1230 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 119-121 and ending at the stop codon at nucleotide positions1385-1387 (FIG. 13). The predicted polypeptide precursor is 422 aminoacids long (FIG. 14). The full-length PRO327 protein shown in FIG. 14has an estimated molecular weight of about 46,302 daltons and a pI ofabout 9.42. Clone DNA38113-1230 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209530.

[0530] Analysis of the amino acid sequence of the full-length PRO327polypeptide suggests that it possess significant homology to the humanprolactin receptor protein, thereby indicating that PRO327 may be anovel prolactin binding protein.

Example 9 Isolation of cDNA Clones Encoding Human PRO233

[0531] A consensus DNA sequence was assembled relative to other ESTsequences as described in Example 1 above. This consensus sequence isherein designated DNA30945. Based on the DNA30945 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO233.

[0532] PCR primers were synthesized as followed: forward PCR primer5′-GGTGAAGGCAGAAATTGGAGATG-3′ (SEQ ID NO:38) reverse PCR primer5′-ATCCCATGCATCAGCCTGTTTACC-3′ (SEQ ID NO:39)

[0533] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30945 sequence which had the followingnucleotide sequence hybridization probe5′-GCTGGTGTAGTCTATACATCAGATTTGTTTGCTACACAAGATCCTCAG-3′ (SEQ ID NO:40)

[0534] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pair identified above. A positivelibrary was then used to isolate clones encoding the PRO233 gene usingthe probe oligonucleotide. RNA for construction of the cDNA librarieswas isolated from human fetal brain tissue.

[0535] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO233 [herein designated as DNA34436-1238](SEQ ID NO:36) and the derived protein sequence for PRO233.

[0536] The entire nucleotide sequence of DNA34436-1238 is shown in FIG.15 (SEQ ID NO:36). Clone DNA34436-1238 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 101-103 and ending at the stop codon at nucleotide positions1001-1003 (FIG. 15). The predicted polypeptide precursor is 300 aminoacids long (FIG. 16). The full-length PRO233 protein shown in FIG. 16has an estimated molecular weight of about 32,964 daltons and a pI ofabout 9.52. In addition, regions of interest including the signalpeptide and a putative oxidoreductase active site, are designated inFIG. 16. Clone DNA34436-1238 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209523

[0537] Analysis of the amino acid sequence of the full-length PRO233polypeptide suggests that portions of it possess significant homology tovarious reductase proteins, thereby indicating that PRO233 may be anovel reductase.

Example 10 Isolation of cDNA Clones Encoding Human PRO344

[0538] A consensus DNA sequence was assembled relative to other ESTsequences as described in Example 1 above. This consensus sequence isherein designated DNA34398. Based on the DNA34398 consensus sequencs,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO344.

[0539] Based on the DNA34398 consensus sequence, forward and reverse PCRprimers were synthesized as follows: forward PCR primer (34398.f1)5′-TACAGGCCCAGTCAGGACCAGGGG-3′ (SEQ ID NO:43) forward PCR primer(34398.f2) 5′-AGCCAGCCTCGCTCTCGG-3′ (SEQ ID NO:44) forward PCR primer(34398.f3) 5′-GTCTGCGATCAGGTCTGG-3′ (SEQ ID NO:45) reverse PCR primer(34398.r1) 5′-GAAAGAGGCAATGGATTCGC-3′ (SEQ ID NO:46) reverse PCR primer(34398.r2) 5′-GACTTACACTTGCCAGCACAGCAC-3′ (SEQ ID NO:47)

[0540] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA34398 consensus sequence which had the followingnucleotide sequence hybridization probe (34398.p1)5′-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3′ (SEQ ID NO:48)

[0541] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with one of the PCR primer pairs identified above. Apositive library was then used to isolate clones encoding the PRO344genes using the probe oligonucleotide and one of the PCR primers. RNAfor construction of the cDNA libraries was isolated from human fetalkidney tissue.

[0542] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO344 [herein designated as DNA40592-1242](SEQ ID NO:41) and the derived protein sequence for PRO344.

[0543] The entire nucleotide sequence of DNA40592-1242 is shown in FIG.17 (SEQ ID NO:41). Clone DNA40592-1242 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 227-229 and ending at the stop codon at nucleotide positions956-958 (FIG. 17). The predicted polypeptide precursor is 243 aminoacids long (FIG. 18). Important regions of the native PRO344 amino acidsequence include the signal peptide, the start of the mature protein,and two potential N-myristoylation sites as shown in FIG. 18. CloneDNA40592-1242 has been deposited with the ATCC and is assigned ATCCdeposit no. ATCC 209492

[0544] Analysis of the amino acid sequence of the full-length PRO344polypeptides suggests that portions of them possess significant homologyto various human and murine complement proteins, thereby indicating thatPRO344 may be a novel complement protein.

Example 11 Isolation of cDNA Clones Encoding Human PRO347

[0545] A consensus DNA sequence was assembled relative to other ESTsequences as described in Example 1 above. This consensus sequence isherein designated DNA39499. Based on the DNA39499 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO347.

[0546] PCR primers (forward and reverse) were synthesized as follows:forward PCR primer 5′-AGGAACTTCTGGATCGGGCTCACC-3′ (SEQ ID NO:51) reversePCR primer 5′-GGGTCTGGGCCAGGTGGAAGAGAG-3′ (SEQ ID NO:52)

[0547] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA39499 sequence which had the followingnucleotide sequence hybridization probe5′-GCCAAGGACTCCTTCCGCTGGGCCACAGGGGAGCACCAGGCCTTC-3′ (SEQ ID NO:53)

[0548] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pair identified above. A positivelibrary was then used to isolate clones encoding the PRO347 gene usingthe probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal kidneytissue (LIB228).

[0549] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO347 [herein designated as DNA44176-1244](SEQ ID NO:49) and the derived protein sequence for PRO347.

[0550] The entire nucleotide sequence of DNA44176-1244 is shown in FIG.19 (SEQ ID NO:49). Clone DNA44176-1244 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 123-125 and ending at the stop codon at nucleotide positions1488-1490 (FIG. 19). The predicted polypeptide precursor is 455 aminoacids long (FIG. 20). The full-length PRO347 protein shown in FIG. 20has an estimated molecular weight of about 50,478 daltons and a pI ofabout 8.44. Clone DNA44176-1244 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209532

[0551] Analysis of the amino acid sequence of the full-length PRO347polypeptide suggests that portions of it possess significant homology tovarious cysteine-rich secretory proteins, thereby indicating that PRO347may be a novel cysteine-rich secretory protein.

Example 12 Isolation of cDNA Clones Encoding Human PRO354

[0552] An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and various ESTsequences were identified which possessed certain degress of homologywith the inter-alpha-trypsin inhibitor heavy chain and with one another.Those homologous EST sequences were then aligned and a consensussequence was obtained. The obtained consensus DNA sequence was thenextended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using homologous EST sequencesderived from both public EST databases (e.g., GenBank) and a proprietaryEST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, Calif.).The extended assembly sequence is herein designated DNA39633. The abovesearches were performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

[0553] Based on the DNA39633 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO354. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

[0554] PCR primers were synthesized as follows: forward PCR primer 1(39633.f1) 5′-GTGGGAACCAAACTCCGGCAGACC-3′ (SEQ ID NO:56) forward PCRprimer 2 (39633.f2) 5′-CACATCGAGCGTCTCTGG-3′ (SEQ ID NO:57) reverse PCRprimer (39633.r1) 5′-AGCCGCTCCTTCTCCGGTTCATCG-3′ (SEQ ID NO:58)

[0555] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA39633 sequence which had the followingnucleotide sequence hybridization probe5′-TGGAAGGACCACTTGATATCAGTCACTCCAGACAGCATCAGGGATGGG-3′ (SEQ ID NO:59)

[0556] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with the PCR primer pairs identified above. A positivelibrary was then used to isolate clones encoding the PRO354 gene usingthe probe oligonucleotide and one of the PCR primers.

[0557] RNA for construction of the cDNA libraries was isolated fromhuman fetal kidney tissue (LIB227). The cDNA libraries used to isolatethe cDNA clones were constructed by standard methods using commerciallyavailable reagents such as those from Invitrogen, San Diego, Calif. ThecDNA was primed with oligo dT containing a NotI site, linked with bluntto SalI hemikinased adaptors, cleaved with NotI, sized appropriately bygel electrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

[0558] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO354 [herein designated as DNA44192-1246](SEQ ID NO:54) and the derived protein sequence for PRO354.

[0559] The entire nucleotide sequence of DNA44192-1246 is shown in FIG.21 (SEQ ID NO:54). Clone DNA44192-1246 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 72-74 and ending at the stop codon at nucleotide positions2154-2156 (FIG. 21). The predicted polypeptide precursor is 694 aminoacids long (FIG. 22). The full-length PRO354 protein shown in FIG. 22has an estimated molecular weight of about 77,400 daltons and a pI ofabout 9.54. Clone DNA44192-1246 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209531.

[0560] Analysis of the amino acid sequence of the full-length PRO354polypeptide suggests that it possess significant homology to theinter-alpha-trypsin inhibitor heavy chain protein, thereby indicatingthat PRO354 may be a novel inter-alpha-trypsin inhibitor heavy chainprotein homolog.

Example 13 Isolation of cDNA Clones Encoding Human PRO355

[0561] A consensus DNA sequence was assembled relative to other ESTsequences using BLAST and phrap as described in Example 1 above. Thisconsensus sequence is herein designated DNA35702. Based on the DNA35702consensus sequence, oligonucleotides were synthesized: 1) to identify byPCR a cDNA library that contained the sequence of interest, and 2) foruse as probes to isolate a clone of the full-length coding sequence forPRO355.

[0562] Forward and reverse PCR primers were synthesized as follows:forward PCR primer 5′-GGCTTCTGCTGTTGCTCTTCTCCG-3′ (SEQ ID NO:62) forwardPCR primer 5′-GTACACTGTGACCAGTCAGC-3′ (SEQ ID NO:63) forward PCR primer5′-ATCATCACAGATTCCCGAGC-3′ (SEQ ID NO:64) reverse PCR primer5′-TTCAATCTCCTCACCTTCCACCGC-3′ (SEQ ID NO:65) reverse PCR primer5′-ATAGCTGTGTCTGCGTCTGCTGCG-3′ (SEQ ID NO:66)

[0563] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35702 sequence which had the followingnucleotide sequence: hybridization probe5′-CGCGGCACTGATCCCCACAGGTGATGGGCAGAATCTGTTTACGAAAGACG-3′ (SEQ ID NO:67)

[0564] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with one of the PCR primer pairs identified above. Apositive library was then used to isolate clones encoding the PRO355gene using the probe oligonucleotide. RNA for construction of the cDNAlibraries was isolated from human fetal liver tissue.

[0565] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO355 [herein designated as DNA39518-1247](SEQ ID NO:60) and the derived protein sequence for PRO355.

[0566] The entire nucleotide sequence of DNA39518-1247 is shown in FIG.23 (SEQ ID NO:60). Clone DNA39518-1247 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 22-24 and ending at the stop codon at nucleotide positions1342-1344 (FIG. 23). The predicted polypeptide precursor is 440 aminoacids long (FIG. 24). The full-length PRO355 protein shown in FIG. 24has an estimated molecular weight of about 48,240 daltons and a pI ofabout 4.93. In addition, regions of interest including the signalpeptide, Ig repeats in the extracellular domain, potentialN-glycosylation sites, and the potential transmembrane domain, aredesignated in FIG. 24. Clone DNA39518-1247 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209529.

[0567] Analysis of the amino acid sequence of the full-length PRO355polypeptide suggests that portions of it possess significant homology tothe CRTAM protein, thereby indicating that PRO355 may be CRTAM protein.

Example 14 Isolation of cDNA Clones Encoding Human PRO357

[0568] The sequence expression tag clone no. “2452972” by IncytePharmaceuticals, Palo Alto, Calif. was used to begin a data base search.The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases which overlapped with a portion of IncyteEST clone no. “2452972”. The EST databases included public EST databases(e.g., GenBank) and a proprietary EST DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6frame translation of the EST sequence. Those comparisons resulting in aBLAST score of 70 (or in some cases 90) or greater that did not encodeknown proteins were clustered and assembled into consensus DNA sequenceswith the program “phrap” (Phil Green, University of Washington, Seattle,Wash.).

[0569] A consensus DNA sequence was then assembled relative to other ESTsequences using phrap. This consensus sequence is herein designatedDNA37162. In this case, the consensus DNA sequence was extended usingrepeated cycles of BLAST and phrap to extend the consensus sequence asfar as possible using the sources of EST sequences discussed above.

[0570] Based on the DNA37162 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO357. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as ber Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

[0571] PCR primers were synthesized as follows: forward primer 1:5′-CCCTCCACTGCCCCACCGACTG-3′ (SEQ ID NO:70); reverse primer 1:5′-CGGTTCTGGGGACGTTAGGGCTCG-3′ (SEQ ID NO:71); and forward primer 2:5′-CTGCCCACCGTCCACCTGCCTCAAT-3′ (SEQ ID NO:72).

[0572] Additionally, two synthetic oligonucleotide hybridization probeswere constructed from the consensus DNA37162 sequence which had thefollowing nucleotide sequences: hybridization probe 1:5′-AGGACTGCCCACCGTCCACCTGCCTCAATGGGGGCACATGCCACC-3′ (SEQ ID NO:73); andhybridization probe 2:5′-ACGCAAAGCCCTACATCTAAGCCAGAGAGAGACAGGGCAGCTGGG-3′ (SEQ ID NO:74).

[0573] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with a PCR primer pair identified above. A positivelibrary was then used to isolate clones encoding the PRO357 gene usingthe probe oligonucleotide and one of the PCR primers.

[0574] RNA for construction of the cDNA libraries was isolated fromhuman fetal liver tissue. The cDNA libraries used to isolate the cDNAclones were constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

[0575] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO357 [herein designated as DNA44804-1248](SEQ ID NO:68) and the derived protein sequence for PRO357.

[0576] The entire nucleotide sequence of DNA44804-1248 is shown in FIG.25 (SEQ ID NO:68). Clone DNA44804-1248 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 137-139 and ending at the stop codon at nucleotide positions1931-1933 (FIG. 25). The predicted polypeptide precursor is 598 aminoacids long (FIG. 26). Clone DNA44804-1248 has been deposited with ATCCand is assigned ATCC deposit no. ATCC 209527.

[0577] Analysis of the amino acid sequence of the full-length PRO357polypeptide therefore suggests that portions of it possess significanthomology to ALS, thereby indicating that PRO357 may be a novel leucinerich repeat protein related to ALS.

Example 15 Isolation of cDNA Clones Encoding Human PRO715

[0578] A proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals,Palo Alto, Calif.) was searched for EST sequences encoding polypeptideshaving homology to human TNF-α. This search resulted in theidentification of Incyte Expressed Sequence Tag No. 2099855.

[0579] A consensus DNA sequence was then assembled relative to other ESTsequences using seqext and “phrap” (Phil Green, University ofWashington, Seattle, Wash.). This consensus sequence is hereindesignated DNA52092. Based upon the alignment of the various EST clonesidentified in this assembly, a single EST clone from theMerck/Washington University EST set (EST clone no. 725887, Accession No.AA292358) was obtained and its insert sequenced. The full-lengthDNA52722-1229 sequence was then obtained from sequencing the insert DNAfrom EST clone no. 725887.

[0580] The entire nucleotide sequence of DNA52722-1229 is shown in FIG.27 (SEQ ID NO:75). Clone DNA52722-1229 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 114-116 and ending at the stop codon at nucleotide positions864-866 (FIG. 27). The predicted polypeptide is 250 amino acids long(FIG. 28). The full-length PRO715 protein shown in FIG. 28 has anestimated molecular weight of about 27,433 daltons and a pI of about9.85.

[0581] Analysis of the amino acid sequence of the full-length PRO715polypeptide suggests that it possesses significant homology to membersof the tumor necrosis factor family of proteins, thereby indicating thatPRO715 is a novel tumor necrosis factor protein.

Example 16

[0582] Isolation of cDNA Clones Encoding Human PRO353

[0583] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1 above. This consensussequences is herein designated DNA36363. The consensus DNA sequence wasextended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using the sources of EST sequencesdiscussed above. Based on the DNA36363 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO353.

[0584] Based on the DNA36363 consensus sequence, forward and reverse PCRprimers were synthesized as follows: forward PCR primer5′-TACAGGCCCAGTCAGGACCAGGGG-3′ (SEQ ID NO:79) reverse PCR primer5′-CTGAAGAAGTAGAGGCCGGGCACG-3′ (SEQ ID NO:80).

[0585] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA36363 consensus sequence which had the followingnucleotide sequence: hybridization probe5′-CCCGGTGCTTGCGCTGCTGTGACCCCGGTACCTCCATGTACCCGG-3′ (SEQ ID NO:81)

[0586] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with one of the PCR primer pairs identified above. Apositive library was then used to isolate clones encoding the PRO353gene using the probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal kidneytissue.

[0587] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO353 [herein designated as DNA41234-1242](SEQ ID NO:77) and the derived protein sequence for PRO353.

[0588] The entire nucleotide sequence of DNA41234-1242 is shown in FIG.29 (SEQ ID NO:77). Clone DNA41234-1242 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 305-307 and ending at the stop codon at nucleotide positions1148-1150 (FIG. 29). The predicted polypeptide precursor is 281 aminoacids long (FIG. 30). Important regions of the amino acid sequenceencoded by PRO353 include the signal peptide, corresponding to aminoacids 1-26, the start of the mature protein at amino acid position 27, apotential N-glycosylation site, corresponding to amino acids 93-98 and aregion which has homology to a 30 kd adipocyte complement-relatedprotein precursor, corresponding to amino acids 99-281. CloneDNA41234-1242 has been deposited with the ATCC and is assigned ATCCdeposit no. ATCC 209618.

[0589] Analysis of the amino acid sequence of the full-length PRO353polypeptides suggests that portions of them possess significant homologyto portions of human and murine complement proteins, thereby indicatingthat PRO353 may be a novel complement protein.

Example 17 Isolation of cDNA Clones Encoding Human PRO361

[0590] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example I above. This consensussequence is herein designated DNA40654. Based on the DNA40654 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO361.

[0591] Forward and reverse PCR primers were synthesized as follows:forward PCR primer 5′-AGGGAGGATTATCCTTGACCTTTGAAGACC-3′ (SEQ ID NO:84)forward PCR primer 5′-GAAGCAAGTGCCCAGCTC-3′ (SEQ ID NO:85)forward PCR primer 5′-CGGGTCCCTGCTCTTTGG-3′ (SEQ ID NO:86)reverse PCR primer 5′-CACCGTAGCTGGGAGCGCACTCAC-3′ (SEQ ID NO:87)reverse PCR primer 5′-AGTGTAAGTCAAGCTCCC-3′ (SEQ ID NO:88)

[0592] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA40654 sequence which had the followingnucleotide sequence hybridization probe5′-GCTTCCTGACACTAAGGCTGTCTGCTAGTCAGAATTGCCTCAAAAAGAG-3′ (SEQ ID NO:89)

[0593] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with one of the PCR primer pairs identified above. Apositive library was then used to isolate clones encoding the PRO361gene using the probe oligonucleotide. RNA for construction of the cDNAlibraries was isolated from human fetal kidney tissue.

[0594] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO361 [herein designated as DNA45410-1250](SEQ ID NO:82) and the derived protein sequence for PRO361.

[0595] The entire nucleotide sequence of DNA45410-1250 is shown in FIG.31 (SEQ ID NO:82). Clone DNA45410-1250 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 226-228 and ending at the stop codon at nucleotide positions1519-1521 (FIG. 31). The predicted polypeptide precursor is 431 aminoacids long (FIG. 32). The full-length PRO361 protein shown in FIG. 32has an estimated molecular weight of about 46,810 daltons and a pI ofabout 6.45. In addition, regions of interest including the transmembranedomain (amino acids 380-409) and sequences typical of the arginasefamily of proteins (amino acids 3-14 and 39-57) are designated in FIG.32. Clone DNA45410-1250 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209621.

[0596] Analysis of the amino acid sequence of the full-length PRO361polypeptide suggests that portions of it possess significant homology tothe mucin and/or chitinase proteins, thereby indicating that PRO361 maybe a novel mucin and/or chitinase protein.

Example 18 Isolation of cDNA Clones Encoding Human PRO365

[0597] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1 above. This consensussequence is herein designated DNA35613. Based on the DNA35613 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO365.

[0598] Forward and reverse PCR primers were synthesized as follows:forward PCR primer 5′-AATGTGACCACTGGACTCCC-3′ (SEQ ID NO:92)forward PCR primer 5′-AGGCTTGGAACTCCCTTC-3′ (SEQ ID NO:93)reverse PCR primer 5′-AAGATTCTTGAGCGATTCCAGCTG-3′ (SEQ ID NO:94)

[0599] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35613 sequence which had the followingnucleotide sequence hybridization probe5′-AATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACTG-3′ (SEQ ID NO:95)

[0600] In order to screen several libraries for a source of afull-length clone, DNA from the libraries was screened by PCRamplification with one of the PCR primer pairs identified above. Apositive library was then used to isolate clones encoding the PRO365gene using the probe oligonucleotide and one of the PCR primers. RNA forconstruction of the cDNA libraries was isolated from human fetal kidneytissue.

[0601] DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO365 [herein designated as DNA46777-1253](SEQ ID NO:90) and the derived protein sequence for PRO365.

[0602] The entire nucleotide sequence of DNA46777-1253 is shown in FIG.33 (SEQ ID NO:90). Clone DNA46777-1253 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 15-17 and ending at the stop codon at nucleotide positions720-722 (FIG. 33). The predicted polypeptide precursor is 235 aminoacids long (FIG. 34). Important regions of the polypeptide sequenceencoded by clone DNA46777-1253 have been identified and include thefollowing: a signal peptide corresponding to amino acids 1-20, the startof the mature protein corresponding to amino acid 21, and multiplepotential N-glycosylation sites as shown in FIG. 34. Clone DNA46777-1253has been deposited with ATCC and is assigned ATCC deposit no. ATCC209619.

[0603] Analysis of the amino acid sequence of the full-length PRO365polypeptide suggests that portions of it possess significant homology tothe human 2-19 protein, thereby indicating that PRO365 may be a novelhuman 2-19 protein homolog.

Example 19 Use of PRO Polypeptide-Encoding Nucleic Acid as HybridizationProbes

[0604] The following method describes use of a nucleotide sequenceencoding PRO as a hybridization probe.

[0605] DNA comprising the coding sequence of full-length or mature PROas disclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

[0606] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled PRO-derived probe to the filters isperformed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodiumpyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution,and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filtersis performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42°C.

[0607] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 20 Expression of PRO Polypeptides in E. coli

[0608] This example illustrates preparation of an unglycosylated form ofPRO by recombinant expression in E. coli.

[0609] The DNA sequence encoding PRO is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene. 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO coding region, lambdatranscriptional terminator, and an argU gene.

[0610] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0611] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0612] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

[0613] PRO may be expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO is initially amplifiedusing selected PCR primers. The primers will contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate .2H20, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

[0614]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

[0615] The proteins are refolded by diluting the sample slowly intofreshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA.Refolding volumes are chosen so that the final protein concentration isbetween 50 to 100 micrograms/ml. The refolding solution is stirredgently at 4° C. for 12-36 hours. The refolding reaction is quenched bythe addition of TFA to a final concentration of 0.4% (pH ofapproximately 3). Before further purification of the protein, thesolution is filtered through a 0.22 micron filter and acetonitrile isadded to 2-10% final concentration. The refolded protein ischromatographed on a Poros RI/H reversed phase column using a mobilebuffer of 0.1% TFA with elution with a gradient of acetonitrile from 10to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDSpolyacrylamide gels and fractions containing homogeneous refoldedprotein are pooled. Generally, the properly refolded species of mostproteins are eluted at the lowest concentrations of acetonitrile sincethose species are the most compact with their hydrophobic interiorsshielded from interaction with the reversed phase resin. Aggregatedspecies are usually eluted at higher acetonitrile concentrations. Inaddition to resolving misfolded forms of proteins from the desired form,the reversed phase step also removes endotoxin from the samples.

[0616] Fractions containing the desired folded PRO polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

[0617] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 21 Expression of PRO Polypeptides in Mammalian Cells

[0618] This example illustrates preparation of a potentiallyglycosylated form of PRO by recombinant expression in mammalian cells.

[0619] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of thePRO DNA using ligation methods such as described in Sambrook et al.,sunra. The resulting vector is called pRK5-PRO.

[0620] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

[0621] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

[0622] In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

[0623] In another embodiment, PRO can be expressed in CHO cells. ThepRK5-PRO can be transfected into CHO cells using known reagents such asCaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of PRO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO can then be concentrated and purified byany selected method.

[0624] Epitope-tagged PRO may also be expressed in host CHO cells. ThePRO may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedPRO insert can then be subcloned into a SV40 driven vector containing aselection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

[0625] PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

[0626] Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG 1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

[0627] Following PCR amplification, the respective DNAs are subcloned ina CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allowthe convenient shuttling of cDNA's. The vector used expression in CHOcells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

[0628] Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

[0629] The ampules containing the plasmid DNA are thawed by placementinto water bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

[0630] For the poly-His tagged constructs, the proteins are purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole is addedto the conditioned media to a concentration of 5 mM. The conditionedmedia is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column is washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0631] Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

[0632] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 22 Expression of PRO in Yeast

[0633] The following method describes recombinant expression of PRO inyeast.

[0634] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

[0635] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0636] Recombinant PRO can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

[0637] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 23 Expression of PRO in Baculovirus-Infected Insect Cells

[0638] The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

[0639] The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

[0640] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0641] Expressed poly-his tagged PRO can then be purified, for example,by Ni²⁺-chelate affinity chromatography as follows. Extracts areprepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatetwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO are pooled and dialyzed againstloading buffer.

[0642] Alternatively, purification of the IgG tagged (or Fc tagged) PROcan be performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

[0643] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 24 Preparation of Antibodies that Bind PRO

[0644] This example illustrates preparation of monoclonal antibodieswhich can specifically bind PRO.

[0645] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO, fusion proteins containingPRO, and cells expressing recombinant PRO on the cell surface. Selectionof the immunogen can be made by the skilled artisan without undueexperimentation.

[0646] Mice, such as Balb/c, are immunized with the PRO immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

[0647] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0648] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO is within the skill in theart.

[0649] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 25 Purification of PRO Polypeptides Using Specific Antibodies

[0650] Native or recombinant PRO polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-PRO polypeptide, mature PRO polypeptide, or pre-PROpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the PRO polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling the anti-PROpolypeptide antibody to an activated chromatographic resin.

[0651] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0652] Such an immunoaffinity column is utilized in the purification ofPRO polypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0653] A soluble PRO polypeptide-containing preparation is passed overthe immunoaffinity column, and the column is washed under conditionsthat allow the preferential absorbance of PRO polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 26 Drug Screening

[0654] This invention is particularly useful for screening compounds byusing PRO polypeptides or binding fragment thereof in any of a varietyof drug screening techniques. The PRO polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

[0655] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect a PRO polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith an PRO polypeptide or fragment thereof and assaying (I) for thepresence of a complex between the agent and the PRO polypeptide orfragment, or (ii) for the presence of a complex between the PROpolypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the PRO polypeptide or fragment istypically labeled. After suitable incubation, free PRO polypeptide orfragment is separated from that present in bound form, and the amount offree or uncomplexed label is a measure of the ability of the particularagent to bind to PRO polypeptide or to interfere with the PROpolypeptide/cell complex.

[0656] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to apolypeptide and is described in detail in WO 84/03564, published on Sep.13, 1984. Briefly stated, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. As applied to a PRO polypeptide, the peptide testcompounds are reacted with PRO polypeptide and washed. Bound PROpolypeptide is detected by methods well known in the art. Purified PROpolypeptide can also be coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies can be used to capture the peptide and immobilize it on thesolid support.

[0657] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 27 Rational Drug Design

[0658] The goal of rational drug design is to produce structural analogsof biologically active polypeptide of interest (i.e., a PRO polypeptide)or of small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19-21 (1991)).

[0659] In one approach, the three-dimensional structure of the PROpolypeptide, or of an PRO polypeptide-inhibitor complex, is determinedby x-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry. 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

[0660] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

[0661] By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 28 Gene Amplification

[0662] This example shows that the PRO327-, PRO344-, PRO347-, PRO357-and PRO715-encoding genes are amplified in the genome of certain humanlung, colon and/or breast cancers and/or cell lines. Amplification isassociated with overexpression of the gene product, indicating that thepolypeptides are useful targets for therapeutic intervention in certaincancers such as colon, lung, breast and other cancers. Therapeuticagents may take the form of antagonists of PRO327, PRO344, PRO347,PRO357 aor PRO715 polypeptide, for example, murine-human chimeric,humanized or human antibodies against a PRO327, PRO344, PRO347, PRO357or PRO715 polypeptide. These amplifications also are useful asdiagnostic markers for the presence of a specific type of tumor type.

[0663] The starting material for the screen was genomic DNA isolatedfrom a variety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm 7700 Sequence Detection System™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding PRO327, PRO344, PRO347, PRO357 orPRO715 is over-represented in any of the primary lung or colon cancersor cancer cell lines or breast cancer cell lines that were screened. Theprimary lung cancers were obtained from individuals with tumors of thetype and stage as indicated in Table 9. An explanation of theabbreviations used for the designation of the primary tumors listed inTable 9 and the primary tumors and cell lines referred to throughoutthis example are given below.

[0664] The results of the TaqMan™ are reported in delta (Δ) Ct units.One unit corresponds to 1 PCR cycle or approximately a 2-foldamplification relative to normal, two units corresponds to 4-fold, 3units to 8-fold amplification and so on. Quantitation was obtained usingprimers and a TaqMan™ fluorescent probe derived from the PRO327-,PRO344-, PRO347-, PRO357- or PRO715-encoding gene. Regions of PRO327,PRO344, PRO347, PRO357 or PRO715 which are most likely to contain uniquenucleic acid sequences and which are least likely to have spliced outintrons are preferred for the primer and probe derivation, e.g.,3′-untranslated regions. The sequences for the primers and probes(forward, reverse and probe) used for the PRO327, PRO344, PRO347, PRO357or PRO715 gene amplification analysis were as follows:PRO327 (DNA38113-1230) forward 5′-CTCAAGAAGCACGCGTACTGC-3′ (SEQ IDNO:96) probe 5′-CCAACCTCAGCTTCCGCCTCTACGA-3′ (SEQ ID NO:97) reverse5′-CATCCAGGCTCGCCACTG-3′ (SEQ ID NO:98)

[0665] PR0344 (DNA40592-1242) forward 5′-TGGCAAGGAATGGGAACAGT-3′ (SEQ IDNO: 99) probe 5′-ATGCTGCCAGACCTGATCGCAGACA-3′ (SEQ ID NO: 100) reverse5′-GGGCAGAAATCCAGCCACT-3′ (SEQ ID NO: 101)

[0666] PR0347 (DNA44176-1244) forward 5′-CCCTTCGCCTGCTTTTGA-3′ (SEQ IDNO: 102) probe 5′-GCCATCTAATTGAAGCCCATCTTCCCA-3′ (SEQ ID NO: 103)reverse 5′-CTGGCGGTGTCCTCTCCTT-3′ (SEQ ID NO: 104)

[0667] PR0357 (DNA44804-1248) forward 5′-CCTCGGTCTCCTCATCTGTGA-3′ (SEQID NO: 105) probe 5′-TGGCCCAGCTGACGAGCCCT-3′ (SEQ ID NO: 106) reverse5′-CTCATAGGCACTCGGTTCTGG-3′ (SEQ ID NO: 107)

[0668] PRO715 (DNA52722-1229) forward 5′-TGGCTCCCAGCTTGGAAGA-3′ (SEQ IDNO: 108) probe 5′-CAGCTCTTGGCTGTCTCCAGTATGTACCCA-3′ (SEQ ID NO: 109)reverse 5′-GATGCCTCTGTTCCTGCACAT-3′ (SEQ ID NO: 110)

[0669] The 5′ nuclease assay reaction is a fluorescent PCR-basedtechnique which makes use of the 5′ exonuclease activity of Taq DNApolymerase enzyme to monitor amplification in real time. Twooligonucleotide primers are used to generate an amplicon typical of aPCR reaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

[0670] The 5′ nuclease procedure is run on a real-time quantitative PCRdevice such as the ABI Prism 7700™ Sequence Detection. The systemconsists of a thermocycler, laser, charge-coupled device (CCD) cameraand computer. The system amplifies samples in a 96-well format on athermocycler. During amplification, laser-induced fluorescent signal iscollected in real-time through fiber optics cables for all 96 wells, anddetected at the CCD. The system includes software for running theinstrument and for analyzing the data.

[0671] 5′ Nuclease assay data are initially expressed as Ct, or thethreshold cycle. This is defined as the cycle at which the reportersignal accumulates above the background level of fluorescence. The ΔCtvalues are used as quantitative measurement of the relative number ofstarting copies of a particular target sequence in a nucleic acid samplewhen comparing cancer DNA results to normal human DNA results.

[0672] Table 9 describes the stage, T stage and N stage of variousprimary tumors which were used to screen the PRO327, PRO344, PRO347,PRO357 and PRO715 compounds of the invention. TABLE 9 Primary Lung andColon Tumor Profiles Primary Tumor Stage Stage Other Stage Dukes Stage TStage N Stage Human lung tumor AdenoCa (SRCC724) [LT1] IIA T1 N1 Humanlung tumor SqCCa (SRCC725) [LT1a] IIB T3 N0 Human lung tumor AdenoCa(SRCC726) [LT2] IB T2 N0 Human lung tumor AdenoCa (SRCC727) [LT3] IIIAT1 N2 Human lung tumor AdenoCa (SRCC728) [LT4] IB T2 N0 Human lung tumorSqCCa (SRCC729) [LT6] IB T2 N0 Human lung tumor Aden/SqCCa (SRCC730)[LT7] IA T1 N0 Human lung tumor AdenoCa (SRCC731) [LT9] IB T2 N0 Humanlung tumor SqCCa (SRCC732) [LT10] IIB T2 N1 Human lung tumor SqCCa(SRCC733) [LT11] IIA T1 N1 Human lung tumor AdenoCa (SRCC734) [LT12] IVT2 N0 Human lung tumor AdenoSqCCa (SRCC735) [LT13] IB T2 N0 Human lungtumor SqCCa (SRCC736) [LT15] IB T2 N0 Human lung tumor SqCCa (SRCC737)[LT16] IB T2 N0 Human lung tumor SqCCa (SRCC738) [LT17] IIB T2 N1 Humanlung tumor SqCCa (SRCC739) [LT18] IB T2 N0 Human lung tumor SqCCa(SRCC740) [LT19] IB T2 N0 Human lung tumor LCCa (SRCC741) [LT21] IIB T3N1 Human lung AdenoCa (SRCC811) [LT22] 1A T1 N0 Human colon AdenoCa(SRCC742) [CT2] M1 D pT4 N0 Human colon AdenoCa (SRCC743) [CT3] B pT3 N0Human colon AdenoCa (SRCC744) [CT8] B T3 N0 Human colon AdenoCa(SRCC745) [CT10] A pT2 N0 Human colon AdenoCa (SRCC746) [CT12] MO, R1 BT3 N0 Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pN0 Human colonAdenoCa (SRCC748) [CT15] M1, R2 D T4 N2 Human colon AdenoCa (SRCC749)[CT16] pMO B pT3 pN0 Human colon AdenoCa (SRCC750) [CT17] C1 pT3 pN1Human colon AdenoCa (SRCC751) [CT1] MO, R1 B pT3 N0 Human colon AdenoCa(SRCC752) [CT4] B pT3 M0 Human colon AdenoCa (SRCC753) [CT5] G2 C1 pT3pN0 Human colon AdenoCa (SRCC754) [CT6] pMO, RO B pT3 pN0 Human colonAdenoCa (SRCC755) [CT7] G1 A pT2 pN0 Human colon AdenoCa (SRCC756) [CT9]G3 D pT4 pN2 Human colon AdenoCa (SRCC757) [CT11] B T3 N0 Human colonAdenoCa (SRCC758) [CT18] MO, RO B pT3 pN0

DNA Preparation

[0673] DNA was prepared from cultured cell lines, primary tumors, normalhuman blood. The isolation was performed using purification kit, bufferset and protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

Cell Culture Lysis

[0674] Cells were washed and trypsinized at a concentration of 7.5×10⁸per tip and pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into 6.2ml cold ddH₂O to a final concentration of 20 mg/ml and equilibrated at4° C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse A stock(100 mg/ml) to a final concentration of 200 μg/ml.

[0675] Buffer C1 (10 ml, 4° C.) and ddH20 (40 ml, 4° C.) were then addedto the 10 ml of cell suspension, mixed by inverting and incubated on icefor 10 minutes. The cell nuclei were pelleted by centrifuging in aBeckman swinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 ml Buffer C1 (at 4° C.) and 6 ml ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30-60 minutes, pelleting at 3000×g for 10 min., 4°C.)

Solid Human Tumor Sample Preparation and Lysis

[0676] Tumor samples were weighed and placed into 50 ml conical tubesand held on ice. Processing was limited to no more than 250 mg tissueper preparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

[0677] Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

Human Blood Preparation and Lysis

[0678] Blood was drawn from healthy volunteers using standard infectiousagent protocols and citrated into 10 ml samples per tip. Quiagenprotease was freshly prepared by dilution into 6.25 ml cold ddH₂O to afinal concentration of 20 mg/ml and stored at 4° C. G2 buffer wasprepared by diluting RNAse A to a final concentration of 200 μg/ml from100 mg/ml stock. The blood (10 ml) was placed into a 50 ml conical tubeand 10 ml C1 buffer and 30 ml ddH₂O (both previously equilibrated to 4°C.) were added, and the components mixed by inverting and held on icefor 10 minutes. The nuclei were pelleted with a Beckman swinging bucketrotor at 2500 rpm, 4° C. for 15 minutes and the supernatant discarded.With a vortex, the nuclei were suspended into 2 ml C 1 buffer (4° C.)and 6 ml ddH₂O (4° C.). Vortexing was repeated until the pellet waswhite. The nuclei were then suspended into the residual buffer using a200 μl tip. G2 buffer (10 ml) were added to the suspended nuclei whilegently vortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

Purification of Cleared Lysates

[0679] (1) Isolation of Genomic DNA

[0680] Genomic DNA was equilibrated (1 sample per maxi tip preparation)with 10 ml QBT buffer. QF elution buffer was equilibrated at 50° C. Thesamples were vortexed for 30 seconds, then loaded onto equilibrated tipsand drained by gravity. The tips were washed with 2×15 ml QC buffer. TheDNA was eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with15 ml QF buffer (50° C.). Isopropanol (10.5 ml) was added to eachsample, the tubes covered with parafin and mixed by repeated inversionuntil the DNA precipitated. Samples were pelleted by centrifugation inthe SS-34 rotor at 15,000 rpm for 10 minutes at 4° C. The pelletlocation was marked, the supernatant discarded, and 10 ml 70% ethanol(4° C.) was added. Samples were pelleted again by centrifugation on theSS-34 rotor at 10,000 rpm for 10 minutes at 4° C. The pellet locationwas marked and the supernatant discarded. The tubes were then placed ontheir side in a drying rack and dried 10 minutes at 37° C., taking carenot to overdry the samples.

[0681] After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5)and placed at 50° C. for 1-2 hours. Samples were held overnight at 4° C.as dissolution continued. The DNA solution was then transferred to 1.5ml tubes with a 26 gauge needle on a tuberculin syringe. The transferwas repeated 5× in order to shear the DNA. Samples were then placed at50°C. for 1-2 hours.

[0682] (2) Quantitation of Genomic DNA and Preparation for GeneAmplification Assay

[0683] The DNA levels in each tube were quantified by standard A₂₆₀,A₂₈₀ spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) usingthe 0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer.A₂₆₀/A₂₈₀ ratios were in the range of 1.8-1.9. Each DNA samples was thendiluted further to approximately 200 ng/ml in TE (pH 8.5). If theoriginal material was highly concentrated (about 700 ng/μl), thematerial was placed at 50° C. for several hours until resuspended.

[0684] Fluorometric DNA quantitation was then performed on the dilutedmaterial (20-600 ng/ml) using the manufacturer's guidelines as modifiedbelow. This was accomplished by allowing a Hoeffer DyNA Quant 200fluorometer to warm-up for about 15 minutes. The Hoechst dye workingsolution (#H33258, 10 μl, prepared within 12 hours of use) was dilutedinto 100 ml 1×TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

[0685] The fluorometricly determined concentration was then used todilute each sample to 10 ng/μl in ddH₂O. This was done simultaneously onall template samples for a single TaqMan plate assay, and with enoughmaterial to run 500-1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8-9 plates or 64 tests.

Gene Amplification Assay

[0686] The PRO327, PRO344, PRO347, PRO357 and PRO715 compounds of theinvention were screened in the following primary tumors and theresulting ΔCt values greater than or equal to 1.0 are reported in Table10. TABLE 10 ΔCt values in primary tumor and cell lines models PrimaryTumors or Cell Lines PRO327 PRO344 PRO347 PRO357 PRO715 LT1 — — 1.035 —1.625 LT1a 1.045 — 1.865 1.18 1.045 1.0 2.47 1.93 LT3 1.135 — 1.325 2.93— 1.2 LT6 1.395 — 1.945 2.6 — 1.42 3.18 LT9 — — 2.645 3.47 1.005 2.91LT10 1.305 — 1.845 3.42 1.125 1.13 3.51 LT11 1.53 1.52 1.395 1.185 1.751.35 2.875 1.12 LT12 2.99 1.2 1.425 1.225 1.63 2.15 1.73 2.225 1.11 1.14LT13 2.48 1.81 2.035 1.585 2.29 1.69 1.175 2.28 1.665 1.83 1.05 1.151.31 LT15 3.99 1.62 1.615 2.205 2.33 2.89 1.33 2.73 2.445 1.89 1.27 1.891.44 LT16 1.16 1.13 — 2.605 1.2 2.65 1.09 1.1 LT17 1.76 1.46 1.24 1.2751.95 1.09 2.855 1.33 1.01 LT18 — — — 2.455 1.14 LT19 3.58 2.47 1.8352.295 2.38 1.35 2.645 LT21 — 1.09 1.14 2.675 — CT2 3.645 1.84 2.1 2.011.675 1.605 1.605 CT3 1.125 — 1.01 — 1.135 1.105 CT8 1.645 — 1.3 1.11.285 1.345 CT10 2.535 — — 1.42 2.155 1.785 CT12 1.885 — — — — CT142.515 1.16 1.39 1.5 1.265 1.45 1.575 CT15 1.305 1.17 1.3 1.25 1.5851.475 CT16 1.475 — 1.33 1.05 1.095 1.055 1.475 CT17 1.715 — — — 1.2451.375 CT1 1.375 1.245 1.045 1.045 1.285 1.6 1.085 CT4 2.225 1.465 —1.275 1.375 2.23 1.165 CT5 2.505 1.515 1.625 1.695 1.975 1.985 2.071.715 CT6 2.285 — — 1.085 1.305 1.73 1.245 CT7 — — — 1.735 1.005 1.651.025 CT9 1.585 — — — 1.0 CT11 3.335 1.355 1.315 1.835 2.185 1.525 2.54CT18 1.075 — — — 1.69 SRCC771 1.65 — — — — (H157) SRCC772 2.23 — — — —(H441) SRCC773 1.12 — — — — (H460) SRCC774 1.18 — — — — (SKMES-1)SRCC777 2.24 — — — — (SW620) SRCC778 1.01 — — — — (Colo320) SRCC830 1.23— — — — (HCC2998) SRCC831 1.61 — — — — (KM12) SRCC832 1.02 — — — —(H522) SRCC833 1.11 — — — — (H810)

[0687] PRO327 (DNA38113-1230) was reexamined along with selected tumorsfrom the above initial screen with framework mapping. Table 11 describesthe framework markers that were employed in association with PRO327(DNA38113-1230). The framework markers are located approximately every20 megabases along Chromosome 19, and are used to control aneuploidy.The ΔCt values for the described framework markers along Chromosome 19relative to PRO327 (DNA38113-1230) are indicated for selected tumors inTable 13.

[0688] PRO327 (DNA38113-1230) was also reexamined along with selectedtumors from the above initial screen with epicenter mapping. Table 12describes the epicenter markers that were employed in association withPRO327 (DNA38113-1230). These markers are located in close proximity toDNA38113-1230 and are used to assess the amplification status of theregion of Chromosome 19 in which DNA38113-1230 is located. The distancebetween markers is measured in centirays (cR), which is a radiationbreakage unit approximately equal to a 1% chance of a breakage betweentwo markers. One cR is very roughly equivalent to 20 kilobases.

[0689] Table 14 indicates the ΔCt values for results of epicentermapping relative to DNA38113-1230, indicating the relative amplificationin the region more immediate to the actual location of DNA38113-1230along Chromosome 19. TABLE 11 Framework Markers Along Chromosome 19 MapPosition on Stanford Human Genome Chromosome 19 Center Marker Name S12 AFMa107xc9 S50  SHGC-31335 S105 SHGC-34102 S155 SHGC-16175

[0690] TABLE 12 Epicenter Markers Along Chromosome 19 used forDNA38113-1230 Map Position Stanford Human Genome Distance to next Markeron Chromosome 19 Center Marker Name (cR¹) S42 WI-7289  5 S43 SHGC-3263828 S44 SHGC-11753²  7 DNA38113-1230 — — S45 SHGC-14810 37 S46 AFM214YF615 S48 SHGC-36583 —

[0691] TABLE 13 Amplification of framework markers relative toDNA38113-1230 (ΔCt) Framework Markers DNA38113- Tumor S12 1230 S50 S105S155 LT1 0.16 −0.15 0.06 −0.42 0.11 LT1a 0.05 0.57 −0.27 0.17 0.40 LT20.48 0.57 0.41 0.52 0.13 LT3 0.27 0.77 0.83 0.11 0.50 LT4 0.48 0.08 0.670.20 0.56 LT6 0.72 0.33 0.74 0.32 0.35 LT7 0.82 0.29 0.85 0.95 0.95 LT90.72 −0.19 0.61 0.19 0.64 LT10 0.82 1.45 0.98 0.62 0.53 CT2 0.25 2.940.29 0.37 −0.02 CT3 −0.17 1.23 −0.10 0.34 −0.28 CT8 0.13 1.45 0.57 0.18−0.16 CT10 0.15 1.72 0.51 −0.01 −0.81 CT12 0.13 1.60 0.57 0.41 0.20 CT140.40 2.03 0.39 0.45 0.36 CT15 −0.23 0.68 −0.30 −0.06 0.56 CT16 0.38 1.070.31 0.24 0.04 CT17 0.25 0.50 0.71 0.32 0.09

[0692] TABLE 14 Amplification of epicenter markers relative toDNA38113-1230 (ΔCt) DNA3811 Tumor S41 S42 S43 S44 3-1230 S45 S46 S48 LT1−1.03 −0.25 −0.18 −0.11 −0.31 0.13 0.26 0.29 LT1a 0.14 −0.30 −0.11 −0.010.21 −0.44 0.45 −0.30 LT2 0.03 0.06 0.06 0.12 0.14 0.16 0.11 0.65 LT3−1.08 −0.08 −0.01 0.11 0.43 −0.37 0.33 0.56 LT4 0.66 −0.14 −0.48 −0.79−0.28 −0.31 0.04 0.09 LT6 −0.88 −0.08 −0.12 −1.00 0.20 −0.43 0.48 0.63LT7 0.65 −0.19 −0.19 −0.04 0.04 −0.42 0.43 0.57 LT9 0.66 −0.26 −0.01−0.14 −0.06 −0.31 −16.48 0.16 LT10 1.16 −0.30 −0.11 −0.31 0.13 −0.330.34 0.50 LT11 0.46 0.01 −0.04 −0.86 0.67 0.23 0.24 −0.57 LT12 1.39−0.01 −0.22 −1.33 1.57 −0.25 0.26 0.07 LT13 1.62 −0.03 0.00 −0.08 1.22−0.08 0.48 0.14 LT15 1.09 0.20 0.47 0.62 2.47 0.38 0.01 0.44 LT16 1.510.04 −0.04 0.29 2.23 0.51 0.50 0.90 LT17 2.12 0.23 0.11 0.20 1.02 0.450.46 −0.41 LT18 1.80 −0.11 0.07 −0.70 0.9 0.10 0.00 −0.02 LT22 −0.120.06 0.41 −0.11 −0.06 0.34 0.03 0.52 CT1 −0.09 0.33 0.11 0.22 1.38 0.09−0.25 −0.10 CT2 1.76 0.04 0.30 0.65 2.94 0.18 −0.04 0.01 CT3 1.10 −0.31−0.24 0.16 1.23 −0.64 0.78 −0.17 CT4 1.63 0.22 0.32 −0.72 2.23 −0.040.44 0.72 CT5 2.22 0.02 0.21 0.10 2.51 0.02 0.18 0.24 CT6 0.48 0.20 0.22−0.63 2.29 0.03 0.14 0.97 CT7 0.93 0.20 0.32 0.14 0.95 −0.01 0.20 0.54CT8 1.15 −0.50 −0.14 0.15 1.45 −0.31 0.54 0.07 CT9 0.82 0.38 0.64 −0.711.59 1.04 0.26 0.93 CT10 1.57 −0.41 −0.03 −0.14 1.72 −0.27 0.04 0.10CT11 1.49 −0.05 0.07 0.01 3.34 0.54 0.28 0.88 CT12 0.89 −0.09 −0.01−0.62 1.6 −0.07 1.16 0.92 CT14 2.16 0.32 0.37 0.47 2.03 −0.07 1.21 0.44CT15 0.64 −0.52 −0.21 −0.12 0.68 −0.61 1.01 0.32 CT16 1.75 −0.31 0.280.47 1.07 0.04 1.01 −0.29 CT17 0.77 −0.18 0.13 −0.04 0.5 −0.27 0.93 0.31CT18 0.91 0.05 0.14 0.60 1.08 0.22 −0.59 0.61

PRO715 (DNA52722-1229)

[0693] PRO715 was also reexamined with both framework and epicentermapping. Table 15 indicates the chromosomal localizations of theframework markers that were used for the procedure. The frameworkmarkers are located approximately every 20 megabases and were used tocontrol aneuploidy. Table 16 indicates the epicenter mapping markersthat were used in the procedure. The epicenter markers were located inclose proximity to DNA52722-1229 and are used to determine the relativeDNA amplification in the immediate vicinity of DNA52722-1229. Thedistance between individual markers is measured in centirays, which is aradiation breakage unit approximately equal to a 1% chance of a breakagebetween two markers. One cR is very roughly equivalent to about 20kilobases. In Table 16, “BAC” means bacterial artificial chromosome. Theends of a BAC clone which contained the gene of interest were sequenced.TaqMan primers and probes were made from this sequence, which areindicated in the table. BAC clones are typically 100 to 150 Kb, so theseprimers and probes can be used as nearby markers to probe DNA fromtumors. In Table 16, the marker SHGC-31370 is the marker found to be theclosest to the location on chromosome 17 where DNA52722-1229 maps. TABLE15 Framework Markers Used Along Chromosome 17 for DNA52722-1229 MapPosition on Stanford Human Genome Chromosome 17 Center Marker Name Q4 SHGC-31242 Q52  SHGC-35988 Q110 AFM200zf4 Q169 SHGC-32689 Q206SHGC-11717 Q232 SHGC-32338

[0694] TABLE 16 Epicenter Markers Used on Chromosome 17 in Vicinity ofDNA52722-1229 Map Position on Stanford Human Genome Distance to nextMarker Chromosome 17 Marker Name (cR) Q33 SHGC-35547 18 cR to Q34120F17FOR1 Marker from forward end of BAC sequence 120F17FOR2 Markerfrom forward end of BAC sequence DNA52722-1229 — 120F17REV1 Marker fromreverse end of BAC sequence 120F17REV2 Marker from reverse end of BACsequence Q34 SHGC-31370

[0695] Table 17 indicates the ΔCt values of the above describedframework markers along chromosome 17 relative to DNA52722-1229 forselected tumors. TABLE 17 Amplification of Framework Markers Relative toDNA52722-1229 Framework Marker DNA5272 Tumor Q4 Q52 2-1229 Q110 Q169Q206 Q232 LT1 0.02 −0.50 −0.04 0.05 −0.32 −0.21 −0.34 LT1a −0.01 −0.340.64 0.23 −0.20 −0.25 −0.15 LT2 0.25 0.15 0.19 0.05 −0.16 −0.14 −0.09LT3 −0.08 −0.20 0.54 0.56 −0.06 0.32 0.05 LT4 −0.32 −0.45 0.31 0.19−0.06 −0.12 0.04 LT6 −0.21 −0.38 0.31 0.13 −0.08 −0.30 0.01 LT7 −0.66−1.02 0.02 0.62 −0.20 0.06 0.16 LT9 −0.03 −0.29 0.46 1.20 −1.75 −0.22−0.13 LT10 −0.16 −0.09 0.58 0.11 0.01 −0.33 −0.45 LT11 −0.14 0.29 1.030.04 0.30 0.52 0.17 LT12 −0.25 −0.68 0.72 0.65 0.86 0.97 0.58 LT13 0.200.00 1.37 −0.15 −0.04 0.25 −0.01 LT15 0.11 −0.39 1.75 0.00 −0.02 0.43−0.19 LT16 −0.07 −0.56 1.11 0.22 0.19 0.68 −0.55 LT17 0.41 −0.09 1.140.27 0.22 0.73 0.07 LT18 0.14 −0.22 1.04 0.27 0.35 0.48 −0.03 LT22 −0.07−0.73 0.00 0.13 −0.02 0.41 0.05 CT2 0.12 −0.47 1.29 −0.19 0.32 — 0.18CT3 0.05 0.17 1.06 −0.41 0.05 — −0.06 CT8 0.44 0.14 1.08 0.02 −0.04 —−0.11 CT10 0.35 0.26 1.60 −0.05 0.00 — −0.02 CT12 −0.15 −0.46 0.52 −0.130.02 — −0.20 CT14 0.26 −0.59 1.05 −0.01 0.68 — 0.48 CT15 0.55 −0.51 1.36−0.69 0.11 — −0.16 CT16 0.09 −0.14 1.06 0.00 0.00 — −0.15 CT17 0.40−0.16 1.00 −0.47 0.04 — −0.29

[0696] Table 18 indicates the ΔCt values for the indicated epicentermarkers, indicating the relative amplification along chromosome 17 inthe immediate vicinity of DNA52722-1229. TABLE 18 Amplification ofEpicenter Markers Relative to DNA52722-1229 Epicenter marker 120F17FOR120F17FOR DNA5272 Tumor Q33 1 2 2-1229 120F17REV1 120F17REV2 Q34 LT1−0.18 0.11 0.00 0.20 −0.08 0.07 −0.36 LT1a 0.32 −0.06 0.00 0.68 −0.09−0.20 0.32 LT2 0.06 0.14 0.00 0.27 −0.29 0.16 −0.16 LT3 0.08 −2.06 0.000.16 −0.84 −0.38 −0.16 LT4 — — — — — — — LT6 — — — — — — — LT7 −0.20−0.51 0.00 0.23 −0.63 −0.37 −0.41 LT9 0.08 −0.17 0.00 0.59 0.02 −0.66−0.01 LT10 0.09 0.05 0.00 0.59 −0.22 −0.12 0.36 LT11 0.75 0.09 0.00 1.070.43 −0.01 0.63 LT12 0.00 −0.45 0.00 0.63 −0.49 −0.82 0.18 LT13 0.72−0.02 0.00 1.29 0.04 0.02 0.66 LT15 0.75 0.11 0.00 1.33 0.15 −0.19 0.90LT16 0.34 −0.41 0.00 1.11 −0.39 −0.89 0.15 LT17 1.06 0.29 0.00 1.13−0.26 −0.12 0.90 LT18 0.66 0.11 0.00 1.21 −0.28 0.11 0.47 LT19 −0.09−0.37 0.00 0.12 −0.53 −0.48 −0.53 CT1 0.50 0.14 0.00 1.22 0.27 0.43 0.72CT2 0.69 −0.47 0.00 0.95 −0.72 −0.17 0.77 CT3 0.87 0.08 0 1.19 −0.060.74 0.97 CT4 0.45 −0.11 0 1.26 0.43 0.38 0.79 CT5 0.36 −0.39 0 1.79−0.48 0.09 0.95 CT6 0.41 0.08 0 1.71 −0.21 0.57 0.47 CT7 0.40 0.18 01.19 0.31 0.40 0.54 CT8 0.48 0.17 0 0.93 0.23 0.47 0.72 CT10 0.72 0.15 01.86 0.81 0.67 0.97 CT11 0.80 −0.09 0 2.29 0.20 0.25 0.85 CT12 0.01−0.55 0 0.49 −0.43 −0.09 0.11 CT14 0.22 −0.36 0 1.05 0.63 0.41 0.40 CT151.06 −0.04 0 1.27 0.74 0.98 1.13 CT16 0.84 0.06 0 1.03 0.26 0.40 0.91CT17 0.80 0.04 0 0.95 0.78 1.29 0.90 CT18 0.34 0.13 0 1.06 0.06 0.340.50

PRO357 (DNA44804-1248)

[0697] PRO357 was reexamined with selected tumors from the above initialscreen with framework mapping. Table 19 indicate the chromosomal mappingof the framework markers that were used in the present example. Theframework markers are located approximately every 20 megabases and wereused to control aneuploidy.

[0698] PRO357 was also examined with epicenter mapping. The markersindicated in Table 20 are located in close proximity (in the genome) toDNA44804-1248 and are used to assess the relative amplification in theimmediate vicinity of chromosome16 wherein DNA44804-1248 is located. Thedistance between individual markers is measured in centirays (cR), whichis a radiation breakage unit approximately equal to a 1% chance of abreakage between the two markers. One cR is very roughly equivalent to20 kilobases. The marker SHGC-6154 is the marker found to be the closestto the location on chromosome 16 where DNA44804-1248 maps. TABLE 19Framework markers for DNA44804-1248 Stanford Human Genome Center Mapposition on chromosome 16 Marker Name P7  SHGC-2835  P55  SHGC-9643 P99  GATA7B02 P154 SHGC-33727 P208 SHGC-13577

[0699] TABLE 20 Epicenter markers for DNA44804-1248 along chromosome 16Map position on Stanford Human Genome Distance to next chromosome 16Center Marker Name Marker (cR) P1 AFMA139WG1  6 P3 SHGC-32420  170 (gap)P4 SHGC-14817 40 P5 SHGC-12265  4 P6 SHGC-6154 33 DNA44804-1248 — — P7SHGC-2835 10 P8 SHGC-2850  9 P9 AFM297yg5 67  P15 CHLC.GATA70B04 —

[0700] The ΔCt values of the above described framework markers alongchromosome 16 relative to DNA44804-1248 is described in Table 21. TABLE21 Amplification of Framework Markers relative to DNA44804-1248 (ΔCt)Framework marker DNA44804- Tumor 1248 P7 P55 P99 P154 208 LT1 0.25 0.22−0.17 0.42 0.04 0.43 LT1a 0.90 0.09 −0.10 −0.38 0.29 0.93 LT2 −0.16 0.030.19 −0.18 0.18 0.54 LT3 1.15 0.68 0.57 −0.34 −0.03 0.86 LT4 0.19 0.580.36 −0.31 0.08 1.14 LT6 0.28 0.27 −0.11 −0.74 −0.13 0.22 LT7 0.58 0.630.14 0.82 0.09 −0.21 LT9 0.68 0.63 0.14 0.82 0.09 −0.21 LT10 1.21 0.520.40 −0.39 −0.15 0.77 LT11 1.71 −0.79 1.31 0.73 −0.08 0.90 LT12 1.96−0.95 0.94 0.00 −0.63 0.18 LT13 2.32 −0.97 0.94 0.88 −0.04 0.70 LT153.01 −0.54 0.60 0.12 0.14 1.15 LT16 0.67 −0.27 0.57 −0.39 0.08 1.04 LT171.64 0.25 1.10 0.28 0.10 0.23 LT18 0.34 0.09 0.51 0.33 −0.20 −0.09 LT193.03 −0.82 0.63 0.06 0.09 0.55 LT21 1.33 −1.19 1.01 0.11 0.34 0.07

[0701] Table 22 indicates the ΔCt values for the results of epicentermapping relative to DNA44804-1248, indicating the relative amplificationin the region more immediate to the actual location of DNA44804-1248along chromosome 16. TABLE 22 Amplification of epicenter markersrelative to DNA44804-1248 Epicenter marker DNA4480 Tumor P1 P3 P4 P5 P64-1248 P7 P8 P9 P15 LT1 0.31 −0.30 0.65 0.05 −0.33 0.16 −0.41 0.20 0.10.17 LT1a −0.23 −17.67 0.97 −0.65 −1.83 0.56 −0.65 −0.28 −0.27 −0.07 LT20.18 −0.06 0.33 −0.11 −0.38 −0.32 −1.08 −0.31 −0.53 −0.05 LT3 0.00 0.251.07 −0.23 −0.11 0.70 −0.71 −0.12 −0.17 −0.01 LT4 0.07 −0.25 0.55 −1.15−1.78 −0.09 −0.82 −0.07 −0.34 −0.07 LT6 0.24 0.07 0.48 −0.55 −0.34 −0.07−1.33 −0.41 −0.7 −0.27 LT7 0.07 −0.07 0.61 −0.19 −0.36 0.29 −0.96 −0.09−0.26 −0.08 LT9 0.16 −0.16 0.64 −0.33 −0.14 0.43 −1.01 −0.19 −0.36 −0.21LT10 0.47 0.76 −0.30 0.80 −0.09 0.00 −0.85 −0.17 −0.28 −0.07 LT11 0.140.14 0.96 −0.02 0.37 1.27 −0.23 0.09 −0.33 −0.07 LT12 −0.12 −0.04 0.84−1.52 −0.28 1.42 −0.39 −0.38 −1.21 −0.25 LT13 0.41 −0.02 1.19 −0.34 0.141.67 −0.87 −0.22 −0.72 −0.33 LT15 0.01 0.21 1.30 −0.48 −0.35 2.36 −0.96−0.36 −0.54 −0.22 LT16 −0.38 −0.07 0.41 −0.32 −1.22 −0.08 −0.45 −0.25−0.52 −0.31 LT17 0.36 0.23 1.39 −1.39 01.37 1.17 −0.39 −0.13 0.52 0.01LT18 0.17 −0.27 0.04 −0.04 0.18 −0.39 −0.59 −0.25 −0.21 −0.22 LT19 0.11−0.02 1.27 −0.12 1.27 2.49 −0.30 −0.36 −0.82 −0.40 LT21 0.28 −0.18 0.850.09 0.66 0.85 −0.49 −0.35 −0.27 −0.16

Conclusion

[0702] The ΔCt values for the above DNAs in a variety of tumors arereported. A ΔCt of >1 was typically used as the threshold value foramplification scoring, as this represents a doubling of gene copy. Theabove data indicates that significant amplification of the testednucleic acids occurred in primary lung tumors and/or primary colontumors: Amplification has been confirmed by framework mapping. Theframework markers analysis reports the relative amplification ofparticular chromosomal regions in the indicated tumors, while theepicenter markers analysis gives a more precise reading of the relativeamplification in the region immediately in the vicinity of the gene ofinterest.

[0703] Amplification has been confirmed by epicenter mapping and thedata evidenced significant amplification in primary colon tumors and/orprimary lung tumors: Amplification of the closest known epicentermarkers does not occur to a greater extent than that of the DNAs tested.This strongly suggests that the DNAs tested are responsible for theamplification of the particular region on the respective chromosome.

[0704] Because amplification of the DNAs tested occurs in various lungand colon tumors, it is highly probable that these DNAs play asignificant role in tumor formation or growth. As a result, antagonists(e.g., antibodies) directed against the proteins encoded by the DNAstested would be expected to have utility in cancer therapy and as usefuldiagnostic reagents. The polypeptides encoded by the DNAs tested haveutility as diagnostic markers for determining the presence of tumorcells in lung and/or colon tissue samples. The nucleic acid sequencesencoding these polypeptides have utility as sources of nucleic acidprobes for carrying out the above diagnostic procedures.

Example 29 Ability of PRO241 to Stimulate the Release of Proteoglycansfrom Cartilage (Assay 97)

[0705] The ability of PRO241 to stimulate the release of proteoglycansfrom cartilage tissue was tested as follows. A positive result in thisassay evidences that the polypeptide is expected to be useful in thetherapeutic treatment of various cartilage and/or bone injuries ordisorders including, for example, arthritis.

[0706] The metacarphophalangeal joint of 4-6 month old pigs wasaseptically dissected, and articular cartilage was removed by free handslicing being careful to avoid the underlying bone. The cartilage wasminced and cultured in bulk for 24 hours in a humidified atmosphere of95% air, 5% CO₂ in serum free (SF) media (DME/F12 1:1) woth 0.1% BSA and100 U/ml penicillin and 100 μg/ml streptomycin. After washing threetimes, approximately 100 mg of articular cartilage was aliquoted intomicronics tubes and incubated for an additional 24 hours in the above SFmedia. PRO241 polypeptides were then added at 1% either alone or incombination with 18 ng/ml interleukin-1α, a known stimulator ofproteoglycan release from cartilage tissue. The supernatant was thenharvested and assayed for the amount of proteoglycans using the1,9-dimethyl-methylene blue (DMB) colorimetric assay (Farndale andButtle, Biochem. Biophys. Acta 883:173-177 (1985)). A positive result inthis assay indicates that the test polypeptide will find use, forexample, in the treatment of sports-related joint problems, articularcartilage defects, osteoarthritis or rheumatoid arthritis. When PRO241polypeptides were tested in the above assay, the polypeptidesdemonstrated a marked ability to stimulate release of proteoglycans fromcartilage tissue both basally and after stimulation with interleukin-1αand at 24 and 72 hours after treatment, thereby indicating that PRO241polypeptides are useful for stimulating proteoglycan release fromcartilage tissue. As such, PRO241 polypeptides are useful for thetreatment of sports-related joint problems, articular cartilage defects,osteoarthritis or rheumatoid arthritis.

Example 30 In Vitro Antitumor Assay with PRO344 (Assay 161)

[0707] The antiproliferative activity of the PRO344 polypeptide wasdetermined in the investigational, disease-oriented in vitro anti-cancerdrug discovery assay of the National Cancer Institute (NCI), using asulforhodamine B (SRB) dye binding assay essentially as described bySkehan et al., J. Natl. Cancer Inst. 82:1107-1112 (1990). The 60 tumorcell lines employed in this study (“the NCI panel”), as well asconditions for their maintenance and culture in vitro have beendescribed by Monks et al., J. Natl. Cancer Inst. 83:757-766(1991). Thepurpose of this screen is to initially evaluate the cytotoxic and/orcytostatic activity of the test compounds against different types oftumors (Monks et al., supra; Boyd, Cancer: Princ. Pract. Oncol. Update3(10):1-12 [1989]).

[0708] Cells from approximately 60 human tumor cell lines were harvestedwith trypsin/EDTA (Gibco), washed once, resuspended in IMEM and theirviability was determined. The cell suspensions were added by pipet (100μL volume) into separate 96-well microtiter plates. The cell density forthe 6-day incubation was less than for the 2-day incubation to preventovergrowth. Inoculates were allowed a preincubation period of 24 hoursat 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100 μL aliquots to themicrotiter plate wells (1:2 dilution). Test compounds were evaluated atfive half-log dilutions (1000 to 100,000-fold). Incubations took placefor two days and six days in a 5% CO₂ atmosphere and 100% humidity.

[0709] After incubation, the medium was removed and the cells were fixedin 0.1 ml of 10% trichloroacetic acid at 40° C. The plates were rinsedfive times with deionized water, dried, stained for 30 minutes with 0.1ml of 0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid,rinsed four times with 1% acetic acid to remove unbound dye, dried, andthe stain was extracted for five minutes with 0.1 ml of 10 mM Tris base[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) ofsulforhodamine B at 492 nm was measured using a computer-interfaced,96-well microtiter plate reader.

[0710] A test sample is considered positive if it shows at least 50%growth inhibitory effect at one or more concentrations. The results areshown in the following Table 23, where the abbreviations are as follows:

[0711] NSCL=non-small cell lung carcinoma

[0712] CNS=central nervous system TABLE 23 Test compound ConcentrationDays Tumor Cell Line Type Cell Line Designation PRO344  1.2 nM 2Leukemia HL-60 (TB) PRO344  1.2 nM 6 Renal UO-31 and CAKI-1 PRO344 14.9nM 2 Colon KM-12 PRO344 14.9 nM 2 CNS SF-268 PRO344 14.9 nM 2 OvarianOVCAR-4 PRO344 14.9 nM 2 Renal CAKI-1 PRO344 14.9 nM 2 Breast MDA-MB-435PRO344 14.9 nM 6 Leukemia HL-60 (TB) PRO344 14.9 nM 6 Colon KM-12 PRO34414.9 nM 6 CNS SF-295 PRO344 14.9 nM 6 NSCL HOP62

[0713] The results of these assays demonstrate that PRO344 polypeptidesare useful for inhibiting neoplastic growth in a number of differenttumor cell types and may be used therapeutically therefor. Antibodiesagainst PRO344 are useful for affinity purification of this usefulpolypeptide. Nucleic acids encoding PRO344 polypeptides are useful forthe recombinant preparation of these polypeptides.

Example31 Inhibition of Vascular Endothelial Growth Factor (VEGF)Stimulated Proliferation of Endothelial Cell Growth (Assay 9)

[0714] The ability of various PRO polypeptides to inhibit VEGFstimulated proliferation of endothelial cells was tested. Polypeptidestesting positive in this assay are useful for inhibiting endothelialcell growth in mammals where such an effect would be beneficial, e.g.,for inhibiting tumor growth.

[0715] Specifically, bovine adrenal cortical capillary endothelial cells(ACE) (from primary culture, maximum of 12-14 passages) were plated in96-well plates at 500 cells/well per 100 microliter. Assay mediaincluded low glucose DMEM, 10% calf serum, 2 mM glutamine, and1×penicillin/streptomycin/fungizone. Control wells included thefollowing: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cellsplus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGFplus 5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in100 microliter volumes), were then added to the wells (at dilutions of1%, 0.1% and 0.01%, respectively). The cell cultures were incubated for6-7 days at 37° C./5% CO₂. After the incubation, the media in the wellswas aspirated, and the cells were washed 1×with PBS. An acid phosphatasereaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1%Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to eachwell. After a 2 hour incubation at 37° C., the reaction was stopped byaddition of 10 microliters 1N NaOH. Optical density (OD) was measured ona microplate reader at 405 nm.

[0716] The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 nm) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1ng/ml, since TGF-beta blocks 70-90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70-90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

[0717] The following polypeptide tested positive in this assay: PRO323.

Example 32 Rod Photoreceptor Cell Survival (Assay 56)

[0718] This assay shows that certain polypeptides of the invention actto enhance the survival/proliferation of rod photoreceptor cells and,therefore, are useful for the therapeutic treatment of retinal disordersor injuries including, for example, treating sight loss in mammals dueto retinitis pigmentosum, AMD, etc. Sprague Dawley rat pups at 7 daypostnatal (mixed population: glia and retinal neuronal cell types) arekilled by decapitation following CO₂ anesthesis and the eyes are removedunder sterile conditions. The neural retina is dissected away form thepigment epithelium and other ocular tissue and then dissociated into asingle cell suspension using 0.25% trypsin in Ca²⁺, Mg²⁺-free PBS. Theretinas are incubated at 37° C. for 7-10 minutes after which the trypsinis inactivated by adding 1 ml soybean trypsin inhibitor. The cells areplated at 100,000 cells per well in 96 well plates in DMEM/F12supplemented with N₂. Cells for all experiments are grown at 37° C. in awater saturated atmosphere of 5% CO₂. After 2-3 days in culture, cellsare fixed using 4% paraformaldehyde, and then stained using CellTrackerGreen CMFDA. Rho 4D2 (ascites or IgG 1:100), a monoclonal antibodydirected towards the visual pigment rhodopsin is used to detect rodphotoreceptor cells by indirect immunofluorescence. The results arecalculated as % survival: total number of calcein-rhodopsin positivecells at 2-3 days in culture, divided by the total number of rhodopsinpositive cells at time 2-3 days in culture. The total cells(fluorescent) are quantified at 20× objective magnification using a CCDcamera and NIH image software for MacIntosh. Fields in the well arechosen at random.

[0719] The following polypeptides tested positive in this assay: PRO243.

Example 33 Pericyte c-Fos Induction (Assay 93)

[0720] This assay shows that certain polypeptides of the invention actto induce the expression of c-fos in pericyte cells and, therefore, areuseful not only as diagnostic markers for particular types ofpericyte-associated tumors but also for giving rise to antagonists whichwould be expected to be useful for the therapeutic treatment ofpericyte-associated tumors. Specifically, on day 1, pericytes arereceived from VEC Technologies and all but 5 ml of media is removed fromflask. On day 2, the pericytes are trypsinized, washed, spun and thenplated onto 96 well plates. On day 7, the media is removed and thepericytes are treated with 100 μl of PRO polypeptide test samples andcontrols (positive control=DME+5% serum+/−PDGF at 500 ng/ml; negativecontrol=protein 32). Replicates are averaged and SD/CV are determined.Fold increase over Protein 32 (buffer control) value indicated bychemiluminescence units (RLU) luminometer reading verses frequency isplotted on a histogram Two-fold above Protein 32 value is consideredpositive for the assay. ASY Matrix: Growth media=low glucose DMEM=20%FBS+1×pen strep+1×fungizone. Assay Media=low glucose DMEM+5% FBS.

[0721] The following polypeptides tested positive in this assay: PRO241.

Example 34 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 67)

[0722] This example shows that one or more of the polypeptides of theinvention are active as inhibitors of the proliferation of stimulatedT-lymphocytes. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an immune response isbeneficial.

[0723] The basic protocol for this assay is described in CurrentProtocols in Immunology, unit 3.12; edited by J. E. Coligan, A. M.Kruisbeek, D. H. Marglies, E. M. Shevach, W Strober, National Insitutesof Health, Published by John Wiley & Sons, Inc.

[0724] More specifically, in one assay variant, peripheral bloodmononuclear cells (PBMC) are isolated from mammalian individuals, forexample a human volunteer, by leukopheresis (one donor will supplystimulator PBMCs, the other donor will supply responder PBMCs). Ifdesired, the cells are frozen in fetal bovine serum and DMSO afterisolation. Frozen cells may be thawed overnight in assay media (37° C.,5% CO₂) and then washed and resuspended to 3×10⁶ cells/ml of assay media(RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine,1% HEPES, 1% non-essential amino acids, 1% pyruvate). The stimulatorPBMCs are prepared by irradiating the cells (about 3000 Rads).

[0725] The assay is prepared by plating in triplicate wells a mixtureof:

[0726] 100:1 of test sample diluted to 1% or to 0.1%,

[0727] 50:1 of irradiated stimulator cells, and

[0728] 50:1 of responder PBMC cells.

[0729] 100 microliters of cell culture media or 100 microliter ofCD4-IgG is used as the control. The wells are then incubated at 37° C.,5% CO₂ for 4 days On day 5, each well is pulsed with tritiated thymidine(1.0 mC/well;Amersham). After 6 hours the cells are washed 3 times andthen the uptake of the label is evaluated.

[0730] In another variant of this assay, PBMCs are isolated from thespleens of Balb/c mice and C57B6 mice. The cells are teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

[0731] Any decreases below control is considered to be a positive resultfor an inhibitory compound, with decreases of less than or equal to 80%being preferred. However, any value less than control indicates aninhibitory effect for the test protein.

[0732] The following polypeptide tested positive in this assay: PRO361.

Example 35 Tissue Expression Distribution

[0733] Oligonucleotide probes were constructed from the PROpolypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200-600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human adult and/or fetal tissuesources and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of the PROpolypeptide-encoding nucleic acid in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe PRO polypeptide-encoding nucleic acid in various different humantissue types provides a diagnostic marker useful for tissue typing, withor without other tissue-specific markers, for determining the primarytissue source of a metastatic tumor, and the like. The results of theseassays are shown in Table 24 below. TABLE 24 Not Significantly NucleicAcid Significantly Expressed In Expressed In DNA34392-1170 liver,kidney, brain, lung placenta DNA39976-1215 brain lung DNA35595-1228pancreas, brain, kidney, liver DNA34436-1238 lung, placenta, braintestis DNA44176-1244 liver brain, lung DNA44192-1246 kidney liverDNA44804-1248 lung, brain DNA41234-1242 lung, liver, kidney brainDNA45410-1250 lung, brain, kidney, liver DNA46777-1253 liver, placenta,brain

Example 36 Fetal Hemoglobin Induction in an Erythroblastic Cell Line(Assay 107)

[0734] This assay is useful for screening PRO polypeptides for theability to induce the switch from adult hemoglobin to fetal hemoglobinin an erythroblastic cell line. Molecules testing positive in this assayare expected to be useful for therapeutically treating various mammalianhemoglobin-associated disorders such as the various thalassemias. Theassay is performed as follows. Erythroblastic cells are plated instandard growth medium at 1000 cells/well in a 96 well format. PROpolypeptides are added to the growth medium at a concentration of 0.2%or 2% and the cells are incubated for 5 days at 37° C. As a positivecontrol, cells are treated with 100 μM hemin and as a negative control,the cells are untreated. After 5 days, cell lysates are prepared andanalyzed for the expression of gamma globin (a fetal marker). A positivein the assay is a gamma globin level at least 2-fold above the negativecontrol.

[0735] The following polypeptide tested positive in this assay: PRO243.

Example 37 In situ Hybridization

[0736] In situ hybridization is a powerful and versatile technique forthe detection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

[0737] In situ hybridization was performed following an optimizedversion of the protocol by Lu and Gillett, Cell Vision 1:169-176 (1994),using PCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [³³-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe synthesis

[0738] 6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol)were speed vac dried. To each tube containing dried ³³P-UTP, thefollowing ingredients were added:

[0739] 2.0 μl 5× transcription buffer

[0740] 1.0 μl DTT (100 mM)

[0741] 2.0 μl NTP mix (2.5 mM: 10 μ; each of 10 mM GTP, CTP & ATP+10 μlH₂O)

[0742] 1.0 μl UTP (50 μM)

[0743] 1.0 μl Rnasin

[0744] 1.0 μl DNA template (1 μg)

[0745] 1.0 μl H2O

[0746] 1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

[0747] The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNasewere added, followed by incubation at 37° C. for 15 minutes. 90 μl TE(10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture waspipetted onto DE81 paper. The remaining solution was loaded in aMicrocon-50 ultrafiltration unit, and spun using program 10 (6 minutes).The filtration unit was inverted over a second tube and spun usingprogram 2 (3 minutes). After the final recovery spin, 100 μl TE wereadded. 1 μl of the final product was pipetted on DE81 paper and countedin 6 ml of Biofluor II.

[0748] The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μlof RNA Mrk III were added to 3 μl of loading buffer. After heating on a95° C. heat block for three minutes, the gel was immediately placed onice. The wells of gel were flushed, the sample loaded, and run at180-250 volts for 45 minutes. The gel was wrapped in saran wrap andexposed to XAR film with an intensifying screen in −70° C. freezer onehour to overnight.

³³P-Hybridization

[0749] A. Pretreatment of Frozen Sections

[0750] The slides were removed from the freezer, placed on aluminiumtrays and thawed at room temperature for 5 minutes. The trays wereplaced in 55° C. incubator for five minutes to reduce condensation. Theslides were fixed for 10 minutes in 4% paraformaldehyde on ice in thefume hood, and washed in 0.5 ×SSC for 5 minutes, at room temperature (25ml 20×SSC+975 ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase Kfor 10 minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

[0751] B. Pretreatment of Paraffin-embedded Sections

[0752] The slides were deparaffinized, placed in SQ H₂O, and rinsedtwice in 2×SSC at room temperature, for 5 minutes each time. Thesections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/mlin 250 ml RNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or8×proteinase K (100 μl in 250 ml Rnas buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

[0753] C. Prehybridization

[0754] The slides were laid out in a plastic box lined with Box buffer(4×SSC, 50% formamide)—saturated filter paper. The tissue was coveredwith 50 μl of hybridization buffer (3.75 g Dextran Sulfate+6 ml SQ H₂O),vortexed and heated in the microwave for 2 minutes with the caploosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC and 9ml SQ H₂O were added, the tissue was vortexed well, and incubated at 42°C. for 14 hours.

[0755] D. Hybridization

[0756] 1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide wereheated at 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

[0757] E. Washes

[0758] Washing was done 2×10 minutes with 2×SSC, EDTA at roomtemperature (400 ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4L), followed byRNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 mlRnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC,EDTA at room temperature. The stringency wash conditions were asfollows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA,V_(f)=4L).

[0759] F. Oligonucleotides

[0760] In situ analysis was performed on a variety of DNA sequencesdisclosed herein. The oligonucleotides employed for these analyses areas follows. (1) DNA44804-1248 (PR0357) p15′-GGATTCTAATACGACTCACTATAGGGCTGCCCGCAACCCCTTCAACTG-3′ (SEQ ID NO:111)p2 5′-CTATGAAATTAACCCTCACTAAAGGGACCGCAGCTGGGTGACCGTGTA-3′ (SEQ IDNO:112) (2) DNA52722-1229 (PR0715) p15′-GGATTCTAATACGACTCACTATAGGGCCGCCCCGCCACCTCCT-3′ (SEQ ID NO:113) p25′-CTATGAAATTAACCCTCACTAAAGGGACTCGAGACACCACCTGACCCA-3′ (SEQ ID NO:114)p3 5′-GGATTCTAATACGACTCACTATAGGGCCCAAGGAAGGCAGGAGACTCT-3′ (SEQ IDNO:115) p4 5′-CTATGAAATTAACCCTCACTAAAGGGACTAGGGGGTGGGAATGAAAAG-3′ (SEQID NO:116)

[0761] (3) DNA38113-1230 (PRO327) p15′-GGATTCTAATACGACTCACTATAGGGCCCCCCTGAGCTCTCCCGTGTA-3′ (SEQ ID NO: 117)p2 5′-CTATGAAATTAACCCTCACTAAAGGGAAGGCTCGCCACTGGTCGTAGA-3′ (SEQ ID NO:118) (4) DNA35917-1207 (PRO243) p15′-GGATTCTAATACGACTCACTATAGGGCAAGGAGCCGGGACCCAGGAGA-3′ (SEQ ID NO: 119)p2 5′-CTATGAAATTAACCCTCACTAAAGGGAGGGGGCCCTTGGTGCTGAGT-3′ (SEQ ID NO:120)

[0762] G. Results

[0763] In situ analysis was performed on a variety of DNA sequencesdisclosed herein. The results from these analyses are as follows.

[0764] (1) DNA44804-1248 (PRO357)

[0765] Low to moderate level expression at sites of bone formation infetal tissues and in the malignant cells of an osteosarcoma. Possiblesignal in placenta and cord. All other tissues negative.

[0766] Fetal tissues examined (E12-E16 weeks) include: liver, kidney,adrenals, lungs, heart, great vessels, oesophagus, stomach, spleen,gonad, brain, spinal cord and body wall.

[0767] Adult human tissues examined: liver, kidney, stomach, spleen,adrenal, pancreas, lung, colonic carcinoma, renal cell carcinoma andosteosarcoma. Acetominophen induced liver injury and hepatic cirrhosis.

[0768] Chimp Tissues examined: thyroid, parathyroid, lymph node, nerve,tongue, thymus, adrenal, gastric mucosa and salivary gland.

[0769] Rhesus Monkey: cerebrum and cerebellum.

[0770] (2) DNA52722-1229 (PRO715)

[0771] Generalized high signal seen over many tissues—highest signalseen over placenta, osteoblasts, injured renal tubules, injured liver,colorectal liver metastasis and gall bladder.

[0772] Fetal tissues examined (E12-E16 weeks) include: placenta,umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, greatvessels, oesophagus, stomach, small intestine, spleen, thymus, pancreas,brain, eye, spinal cord, body wall, pelvis and lower limb.

[0773] Adult human tissues examined: liver, kidney, adrenal, myocardium,aorta, spleen, lung, skin, chondrosarcoma, eye, stomach, colon, coloniccarcinoma, prostate, bladder mucosa and gall bladder. Acetominopheninduced liver injury and hepatic cirrhosis.

[0774] Rhesus Tissues examined: cerebral cortex (rm), hippocampus (rm)

[0775] Chimp Tissues examined: thyroid, parathyroid, lymph node, nerve,tongue, thymus, adrenal, gastric mucosa and salivary gland.

[0776] (3) DNA38113-1230 (PRO327)

[0777] High level of expression observed in developing mouse and humanfetal lung. Normal human adult lung, including bronchial epithelium, wasnegative. Expression in submucosa of human fetal trachea, possibly insmooth muscle cells. Expression also observed in non-trophoblastic cellsof uncertain histogenesis in the human placenta. In the mouse expressionwas observed in the developing snout and in the developing tongue. Allother tissues were negative. Speculated function: Probable role inbronchial development.

[0778] Fetal tissues examined (E12-E16 weeks) include: placenta,umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, greatvessels, oesophagus, stomach, small intestine, spleen, thymus, pancreas,brain, eye, spinal cord, body wall, pelvis and lower limb.

[0779] Adult tissues examined: liver, kidney, adrenal, myocardium,aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),hippocampus (rm), cerebellum (rm), penis, eye, bladder, stomach, gastriccarcinoma, colon, colonic carcinoma, thyroid (chimp), parathyroid(chimp) ovary (chimp) and chondrosarcoma.

[0780] (4) DNA35917-1207 (PRO243)

[0781] Cornelia de Lange syndrome (CdLS) is a congenital syndrome. Thatmeans it is present from birth. CdLS is a disorder that causes a delayin physical, intellectual, and language development. The vast majorityof children with CdLS are mentally retarded, with the degree of mentalretardation ranging from mild to severe. Reported IQ's from 30 to 85.The average IQ is 53. The head and facial features include small headsize, thin eyebrows which often meet at the midline, long eyelashes,short upturned nose, thin downturned lips, lowset ears and high archedpalate or cleft palate. Other characteristics may include languagedelay, even in the most mildly affected, delayed growth and smallstature, low pitched cry, small hands and feet, incurved fifth fingers,simian creases, and excessive body hair. Diagnosis depends on thepresence of a combination of these characteristics. Many of thesecharacteristics appear in varying degrees. In some cases thesecharacteristics may not be present or be so mild that they will berecognized only when observed by a trained geneticist or other personfamiliar with the syndrome. Although much is known about CdLS, recentreports suggest that there is much more to be learned.

[0782] In this study additional sections of human fetal face, head,limbs and mouse embryos were examined. No expression was seen in any ofthe mouse tissues. Expression was only seen with the antisense probe.

[0783] Expression was observed adjacent to developing limb and facialbones in the perosteal mesenchyme. The expression was highly specificand was often adjacent to areas undergoing vascularization. Thedistribution is consistent with the observed skeletal abnormalities inthe Cornelia de Lange syndrome. Expression was also observed in thedeveloping temporal and occipital lobes of the fetal brain, but was notobserved elsewhere. In addition, expression was seen in the ganglia ofthe developing inner ear; the significance of this finding is unclear.

[0784] Though these data do not provide functional information, thedistribution is consistent with the sites that are known to be affectedmost severely in this syndrome.

[0785] Additionally, faint expression was observed at the cleavage linein the developing synovial joint forming between the femoral head andacetabulum (hip joint). If this pattern of expression were observed atsites of joint formation elsewhere, it might explain the facial and limbabnormalities observed in the Cornelia de Lange syndrome.

Example 38 Activity of PRO243 mRNA in Xenopus Oocytes

[0786] In order to demonstrate that the human chordin clone(DNA35917-1207) encoding PRO243 is functional and acts in a mannerpredicted by the Xenopus chordin and Drosophila sog genes, supercoiledplasmid DNA from DNA35917-1207 was prepared by Qiagen and used forinjection into Xenopus laevis embryos. Micro-injection of Xenopuschordin mRNA into ventrovegetal blastomeres induces secondary (twinned)axes (Sasai et al., Cell 79:779-790 (1994)) and Drosophila sog alsoinduces a secondary axis when ectopically expresed on the ventral sideof the Xenopus embryo (Holley et al., Nature 376:249-253 (1995) andSchmidt et al., Development 121:4319-4328 (1995)). The ability of sog tofunction in Xenopus ooctyes suggests that the processes involved indorsoventral patterning have been conserved during evolution.

Methods Manipulation of Xenopus Embryos

[0787] Adult female frogs were boosted with 200 I.U. pregnant mare serum3 days before use and with 800 I.U. of human chorionic gonadotropin thenight before injection. Fresh oocytes were squeezed out from femalefrogs the next morning and in vitro fertilization of oocytes wasperformed by mixing oocytes with minced testis from sacrificed malefrogs. Developing embryos were maintained and staged according toNieuwkoop and Faber, Normal Table of Xenopus laevis, N.-H. P. Co., ed.(Amsterdam, 1967).

[0788] Fertilized eggs were dejellied with 2% cysteine (pH 7.8) for 10minutes, washed once with distilled water and transferred to 0.1×MBSwith 5% Ficoll. Fertilized eggs were lined on injection trays in 0.1×MBSwith 5% Ficoll. Two-cell stage developing Xenopus embryos were injectedwith 200 pg of pRK5 containing wild type chordin (DNA35917-1207) or 200pg of pRK5 without an insert as a control. Injected embryos were kept ontrays for another 6 hours, after which they were transferred to 0.1×MBSwith 50 mg/ml gentamycin until reaching Nieukwkoop stage 37-38.

Results

[0789] Injection of human chordin cDNA into single blastomeres resultedin the ventralization of the tadpole. The ventralization of the tadpoleis visible in the shortening and kinking of the tail and the expansionof the cement gland. The ability of human chordin to function as aventralizing agent in Xenopus shows that the protein encoded byDNA35917-1207 is functional and influences dorsal-ventral patterning infrogs and suggests that the processes involved in dorsoventralpatterning have been conserved during evolution, with mechanisms incommon between humans, flies and frogs.

Deposit of Material

[0790] The following materials have been deposited with the AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209, USA (ATCC): Material ATCC Dep. No. Deposit DateDNA34392-1170 ATCC 209526 December 10, 1997 DNA35917-1207 ATCC 209508December 3, 1997 DNA39976-1215 ATCC 209524 December 10, 1997DNA35595-1228 ATCC 209528 December 10, 1997 DNA38113-1230 ATCC 209530December 10, 1997 DNA34436-1238 ATCC 209523 December 10, 1997DNA40592-1242 ATCC 209492 November 21, 1997 DNA44176-1244 ATCC 209532December 10, 1997 DNA44192-1246 ATCC 209531 December 10, 1997DNA39518-1247 ATCC 209529 December 10, 1997 DNA44804-1248 ATCC 209527December 10, 1997 DNA52722-1229 ATCC 209570 January 7, 1998DNA41234-1242 ATCC 209618 February 5, 1998 DNA45410-1250 ATCC 209621February 5, 1998 DNA46777-1253 ATCC 209619 February 5, 1998

[0791] These deposit were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit. The deposits will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC §122 and the Commissioner's rulespursuant thereto (including 37 CFR §1.14 with particular reference to886 OG 638).

[0792] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0793] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1 120 1 2454 DNA Homo Sapien 1 ggactaatct gtgggagcag tttattccagtatcacccag ggtgcagcca 50 caccaggact gtgttgaagg gtgttttttt tcttttaaatgtaatacctc 100 ctcatctttt cttcttacac agtgtctgag aacatttaca ttatagataa150 gtagtacatg gtggataact tctactttta ggaggactac tctcttctga 200cagtcctaga ctggtcttct acactaagac accatgaagg agtatgtgct 250 cctattattcctggctttgt gctctgccaa acccttcttt agcccttcac 300 acatcgcact gaagaatatgatgctgaagg atatggaaga cacagatgat 350 gatgatgatg atgatgatga tgatgatgatgatgaggaca actctctttt 400 tccaacaaga gagccaagaa gccatttttt tccatttgatctgtttccaa 450 tgtgtccatt tggatgtcag tgctattcac gagttgtaca ttgctcagat500 ttaggtttga cctcagtccc aaccaacatt ccatttgata ctcgaatgct 550tgatcttcaa aacaataaaa ttaaggaaat caaagaaaat gattttaaag 600 gactcacttcactttatggt ctgatcctga acaacaacaa gctaacgaag 650 attcacccaa aagcctttctaaccacaaag aagttgcgaa ggctgtatct 700 gtcccacaat caactaagtg aaataccacttaatcttccc aaatcattag 750 cagaactcag aattcatgaa aataaagtta agaaaatacaaaaggacaca 800 ttcaaaggaa tgaatgcttt acacgttttg gaaatgagtg caaaccctct850 tgataataat gggatagagc caggggcatt tgaaggggtg acggtgttcc 900atatcagaat tgcagaagca aaactgacct cagttcctaa aggcttacca 950 ccaactttattggagcttca cttagattat aataaaattt caacagtgga 1000 acttgaggat tttaaacgatacaaagaact acaaaggctg ggcctaggaa 1050 acaacaaaat cacagatatc gaaaatgggagtcttgctaa cataccacgt 1100 gtgagagaaa tacatttgga aaacaataaa ctaaaaaaaatcccttcagg 1150 attaccagag ttgaaatacc tccagataat cttccttcat tctaattcaa1200 ttgcaagagt gggagtaaat gacttctgtc caacagtgcc aaagatgaag 1250aaatctttat acagtgcaat aagtttattc aacaacccgg tgaaatactg 1300 ggaaatgcaacctgcaacat ttcgttgtgt tttgagcaga atgagtgttc 1350 agcttgggaa ctttggaatgtaataattag taattggtaa tgtccattta 1400 atataagatt caaaaatccc tacatttggaatacttgaac tctattaata 1450 atggtagtat tatatataca agcaaatatc tattctcaagtggtaagtcc 1500 actgacttat tttatgacaa gaaatttcaa cggaattttg ccaaactatt1550 gatacataag gggttgagag aaacaagcat ctattgcagt ttcctttttg 1600cgtacaaatg atcttacata aatctcatgc ttgaccattc ctttcttcat 1650 aacaaaaaagtaagatattc ggtatttaac actttgttat caagcacatt 1700 ttaaaaagaa ctgtactgtaaatggaatgc ttgacttagc aaaatttgtg 1750 ctctttcatt tgctgttaga aaaacagaattaacaaagac agtaatgtga 1800 agagtgcatt acactattct tattctttag taacttgggtagtactgtaa 1850 tatttttaat catcttaaag tatgatttga tataatctta ttgaaattac1900 cttatcatgt cttagagccc gtctttatgt ttaaaactaa tttcttaaaa 1950taaagccttc agtaaatgtt cattaccaac ttgataaatg ctactcataa 2000 gagctggtttggggctatag catatgcttt ttttttttta attattacct 2050 gatttaaaaa tctctgtaaaaacgtgtagt gtttcataaa atctgtaact 2100 cgcattttaa tgatccgcta ttataagcttttaatagcat gaaaattgtt 2150 aggctatata acattgccac ttcaactcta aggaatatttttgagatatc 2200 cctttggaag accttgcttg gaagagcctg gacactaaca attctacacc2250 aaattgtctc ttcaaatacg tatggactgg ataactctga gaaacacatc 2300tagtataact gaataagcag agcatcaaat taaacagaca gaaaccgaaa 2350 gctctatataaatgctcaga gttctttatg tatttcttat tggcattcaa 2400 catatgtaaa atcagaaaacagggaaattt tcattaaaaa tattggtttg 2450 aaat 2454 2 379 PRT Homo Sapien 2Met Lys Glu Tyr Val Leu Leu Leu Phe Leu Ala Leu Cys Ser Ala 1 5 10 15Lys Pro Phe Phe Ser Pro Ser His Ile Ala Leu Lys Asn Met Met 20 25 30 LeuLys Asp Met Glu Asp Thr Asp Asp Asp Asp Asp Asp Asp Asp 35 40 45 Asp AspAsp Asp Asp Glu Asp Asn Ser Leu Phe Pro Thr Arg Glu 50 55 60 Pro Arg SerHis Phe Phe Pro Phe Asp Leu Phe Pro Met Cys Pro 65 70 75 Phe Gly Cys GlnCys Tyr Ser Arg Val Val His Cys Ser Asp Leu 80 85 90 Gly Leu Thr Ser ValPro Thr Asn Ile Pro Phe Asp Thr Arg Met 95 100 105 Leu Asp Leu Gln AsnAsn Lys Ile Lys Glu Ile Lys Glu Asn Asp 110 115 120 Phe Lys Gly Leu ThrSer Leu Tyr Gly Leu Ile Leu Asn Asn Asn 125 130 135 Lys Leu Thr Lys IleHis Pro Lys Ala Phe Leu Thr Thr Lys Lys 140 145 150 Leu Arg Arg Leu TyrLeu Ser His Asn Gln Leu Ser Glu Ile Pro 155 160 165 Leu Asn Leu Pro LysSer Leu Ala Glu Leu Arg Ile His Glu Asn 170 175 180 Lys Val Lys Lys IleGln Lys Asp Thr Phe Lys Gly Met Asn Ala 185 190 195 Leu His Val Leu GluMet Ser Ala Asn Pro Leu Asp Asn Asn Gly 200 205 210 Ile Glu Pro Gly AlaPhe Glu Gly Val Thr Val Phe His Ile Arg 215 220 225 Ile Ala Glu Ala LysLeu Thr Ser Val Pro Lys Gly Leu Pro Pro 230 235 240 Thr Leu Leu Glu LeuHis Leu Asp Tyr Asn Lys Ile Ser Thr Val 245 250 255 Glu Leu Glu Asp PheLys Arg Tyr Lys Glu Leu Gln Arg Leu Gly 260 265 270 Leu Gly Asn Asn LysIle Thr Asp Ile Glu Asn Gly Ser Leu Ala 275 280 285 Asn Ile Pro Arg ValArg Glu Ile His Leu Glu Asn Asn Lys Leu 290 295 300 Lys Lys Ile Pro SerGly Leu Pro Glu Leu Lys Tyr Leu Gln Ile 305 310 315 Ile Phe Leu His SerAsn Ser Ile Ala Arg Val Gly Val Asn Asp 320 325 330 Phe Cys Pro Thr ValPro Lys Met Lys Lys Ser Leu Tyr Ser Ala 335 340 345 Ile Ser Leu Phe AsnAsn Pro Val Lys Tyr Trp Glu Met Gln Pro 350 355 360 Ala Thr Phe Arg CysVal Leu Ser Arg Met Ser Val Gln Leu Gly 365 370 375 Asn Phe Gly Met 3 20DNA Artificial Sequence Synthetic Oligonucleotide Probe 3 ggaaatgagtgcaaaccctc 20 4 24 DNA Artificial Sequence Synthetic OligonucleotideProbe 4 tcccaagctg aacactcatt ctgc 24 5 50 DNA Artificial SequenceSynthetic Oligonucleotide Probe 5 gggtgacggt gttccatatc agaattgcagaagcaaaact gacctcagtt 50 6 3441 DNA Homo Sapien 6 cggacgcgtg ggcggacgcgtgggcccgcs gcaccgcccc cggcccggcc 50 ctccgccctc cgcactcgcg cctccctccctccgcccgct cccgcgccct 100 cctccctccc tcctccccag ctgtcccgtt cgcgtcatgccgagcctccc 150 ggccccgccg gccccgctgc tgctcctcgg gctgctgctg ctcggctccc200 ggccggcccg cggcgccggc ccagagcccc ccgtgctgcc catccgttct 250gagaaggagc cgctgcccgt tcggggagcg gcaggctgca ccttcggcgg 300 gaaggtctatgccttggacg agacgtggca cccggaccta gggcagccat 350 tcggggtgat gcgctgcgtgctgtgcgcct gcgaggcgcc tcagtggggt 400 cgccgtacca ggggccctgg cagggtcagctgcaagaaca tcaaaccaga 450 gtgcccaacc ccggcctgtg ggcagccgcg ccagctgccgggacactgct 500 gccagacctg cccccaggag cgcagcagtt cggagcggca gccgagcggc550 ctgtccttcg agtatccgcg ggacccggag catcgcagtt atagcgaccg 600cggggagcca ggcgctgagg agcgggcccg tggtgacggc cacacggact 650 tcgtggcgctgctgacaggg ccgaggtcgc aggcggtggc acgagcccga 700 gtctcgctgc tgcgctctagcctccgcttc tctatctcct acaggcggct 750 ggaccgccct accaggatcc gcttctcagactccaatggc agtgtcctgt 800 ttgagcaccc tgcagccccc acccaagatg gcctggtctgtggggtgtgg 850 cgggcagtgc ctcggttgtc tctgcggctc cttagggcag aacagctgca900 tgtggcactt gtgacactca ctcacccttc aggggaggtc tgggggcctc 950tcatccggca ccgggccctg gctgcagaga ccttcagtgc catcctgact 1000 ctagaaggccccccacagca gggcgtaggg ggcatcaccc tgctcactct 1050 cagtgacaca gaggactccttgcatttttt gctgctcttc cgagggctgc 1100 tggaacccag gagtggggga ctaacccaggttcccttgag gctccagatt 1150 ctacaccagg ggcagctact gcgagaactt caggccaatgtctcagccca 1200 ggaaccaggc tttgctgagg tgctgcccaa cctgacagtc caggagatgg1250 actggctggt gctgggggag ctgcagatgg ccctggagtg ggcaggcagg 1300ccagggctgc gcatcagtgg acacattgct gccaggaaga gctgcgacgt 1350 cctgcaaagtgtcctttgtg gggctgatgc cctgatccca gtccagacgg 1400 gtgctgccgg ctcagccagcctcacgctgc taggaaatgg ctccctgatc 1450 tatcaggtgc aagtggtagg gacaagcagtgaggtggtgg ccatgacact 1500 ggagaccaag cctcagcgga gggatcagcg cactgtcctgtgccacatgg 1550 ctggactcca gccaggagga cacacggccg tgggtatctg ccctgggctg1600 ggtgcccgag gggctcatat gctgctgcag aatgagctct tcctgaacgt 1650gggcaccaag gacttcccag acggagagct tcgggggcac gtggctgccc 1700 tgccctactgtgggcatagc gcccgccatg acacgctgcc cgtgccccta 1750 gcaggagccc tggtgctaccccctgtgaag agccaagcag cagggcacgc 1800 ctggctttcc ttggataccc actgtcacctgcactatgaa gtgctgctgg 1850 ctgggcttgg tggctcagaa caaggcactg tcactgcccacctccttggg 1900 cctcctggaa cgccagggcc tcggcggctg ctgaagggat tctatggctc1950 agaggcccag ggtgtggtga aggacctgga gccggaactg ctgcggcacc 2000tggcaaaagg catggcctcc ctgatgatca ccaccaaggg tagccccaga 2050 ggggagctccgagggcaggt gcacatagcc aaccaatgtg aggttggcgg 2100 actgcgcctg gaggcggccggggccgaggg ggtgcgggcg ctgggggctc 2150 cggatacagc ctctgctgcg ccgcctgtggtgcctggtct cccggcccta 2200 gcgcccgcca aacctggtgg tcctgggcgg ccccgagaccccaacacatg 2250 cttcttcgag gggcagcagc gcccccacgg ggctcgctgg gcgcccaact2300 acgacccgct ctgctcactc tgcacctgcc agagacgaac ggtgatctgt 2350gacccggtgg tgtgcccacc gcccagctgc ccacacccgg tgcaggctcc 2400 cgaccagtgctgccctgttt gccctgagaa acaagatgtc agagacttgc 2450 cagggctgcc aaggagccgggacccaggag agggctgcta ttttgatggt 2500 gaccggagct ggcgggcagc gggtacgcggtggcaccccg ttgtgccccc 2550 ctttggctta attaagtgtg ctgtctgcac ctgcaaggggggcactggag 2600 aggtgcactg tgagaaggtg cagtgtcccc ggctggcctg tgcccagcct2650 gtgcgtgtca accccaccga ctgctgcaaa cagtgtccag tggggtcggg 2700ggcccacccc cagctggggg accccatgca ggctgatggg ccccggggct 2750 gccgttttgctgggcagtgg ttcccagaga gtcagagctg gcacccctca 2800 gtgccccctt ttggagagatgagctgtatc acctgcagat gtggggcagg 2850 ggtgcctcac tgtgagcggg atgactgttcactgccactg tcctgtggct 2900 cggggaagga gagtcgatgc tgttcccgct gcacggcccaccggcggccc 2950 ccagagacca gaactgatcc agagctggag aaagaagccg aaggctctta3000 gggagcagcc agagggccaa gtgaccaaga ggatggggcc tgagctgggg 3050aaggggtggc atcgaggacc ttcttgcatt ctcctgtggg aagcccagtg 3100 cctttgctcctctgtcctgc ctctactccc acccccacta cctctgggaa 3150 ccacagctcc acaagggggagaggcagctg ggccagaccg aggtcacagc 3200 cactccaagt cctgccctgc caccctcggcctctgtcctg gaagccccac 3250 ccctttcctc ctgtacataa tgtcactggc ttgttgggatttttaattta 3300 tcttcactca gcaccaaggg cccccgacac tccactcctg ctgcccctga3350 gctgagcaga gtcattattg gagagttttg tatttattaa aacatttctt 3400tttcagtcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 3441 7 954 PRT Homo Sapien7 Met Pro Ser Leu Pro Ala Pro Pro Ala Pro Leu Leu Leu Leu Gly 1 5 10 15Leu Leu Leu Leu Gly Ser Arg Pro Ala Arg Gly Ala Gly Pro Glu 20 25 30 ProPro Val Leu Pro Ile Arg Ser Glu Lys Glu Pro Leu Pro Val 35 40 45 Arg GlyAla Ala Gly Cys Thr Phe Gly Gly Lys Val Tyr Ala Leu 50 55 60 Asp Glu ThrTrp His Pro Asp Leu Gly Gln Pro Phe Gly Val Met 65 70 75 Arg Cys Val LeuCys Ala Cys Glu Ala Pro Gln Trp Gly Arg Arg 80 85 90 Thr Arg Gly Pro GlyArg Val Ser Cys Lys Asn Ile Lys Pro Glu 95 100 105 Cys Pro Thr Pro AlaCys Gly Gln Pro Arg Gln Leu Pro Gly His 110 115 120 Cys Cys Gln Thr CysPro Gln Glu Arg Ser Ser Ser Glu Arg Gln 125 130 135 Pro Ser Gly Leu SerPhe Glu Tyr Pro Arg Asp Pro Glu His Arg 140 145 150 Ser Tyr Ser Asp ArgGly Glu Pro Gly Ala Glu Glu Arg Ala Arg 155 160 165 Gly Asp Gly His ThrAsp Phe Val Ala Leu Leu Thr Gly Pro Arg 170 175 180 Ser Gln Ala Val AlaArg Ala Arg Val Ser Leu Leu Arg Ser Ser 185 190 195 Leu Arg Phe Ser IleSer Tyr Arg Arg Leu Asp Arg Pro Thr Arg 200 205 210 Ile Arg Phe Ser AspSer Asn Gly Ser Val Leu Phe Glu His Pro 215 220 225 Ala Ala Pro Thr GlnAsp Gly Leu Val Cys Gly Val Trp Arg Ala 230 235 240 Val Pro Arg Leu SerLeu Arg Leu Leu Arg Ala Glu Gln Leu His 245 250 255 Val Ala Leu Val ThrLeu Thr His Pro Ser Gly Glu Val Trp Gly 260 265 270 Pro Leu Ile Arg HisArg Ala Leu Ala Ala Glu Thr Phe Ser Ala 275 280 285 Ile Leu Thr Leu GluGly Pro Pro Gln Gln Gly Val Gly Gly Ile 290 295 300 Thr Leu Leu Thr LeuSer Asp Thr Glu Asp Ser Leu His Phe Leu 305 310 315 Leu Leu Phe Arg GlyLeu Leu Glu Pro Arg Ser Gly Gly Leu Thr 320 325 330 Gln Val Pro Leu ArgLeu Gln Ile Leu His Gln Gly Gln Leu Leu 335 340 345 Arg Glu Leu Gln AlaAsn Val Ser Ala Gln Glu Pro Gly Phe Ala 350 355 360 Glu Val Leu Pro AsnLeu Thr Val Gln Glu Met Asp Trp Leu Val 365 370 375 Leu Gly Glu Leu GlnMet Ala Leu Glu Trp Ala Gly Arg Pro Gly 380 385 390 Leu Arg Ile Ser GlyHis Ile Ala Ala Arg Lys Ser Cys Asp Val 395 400 405 Leu Gln Ser Val LeuCys Gly Ala Asp Ala Leu Ile Pro Val Gln 410 415 420 Thr Gly Ala Ala GlySer Ala Ser Leu Thr Leu Leu Gly Asn Gly 425 430 435 Ser Leu Ile Tyr GlnVal Gln Val Val Gly Thr Ser Ser Glu Val 440 445 450 Val Ala Met Thr LeuGlu Thr Lys Pro Gln Arg Arg Asp Gln Arg 455 460 465 Thr Val Leu Cys HisMet Ala Gly Leu Gln Pro Gly Gly His Thr 470 475 480 Ala Val Gly Ile CysPro Gly Leu Gly Ala Arg Gly Ala His Met 485 490 495 Leu Leu Gln Asn GluLeu Phe Leu Asn Val Gly Thr Lys Asp Phe 500 505 510 Pro Asp Gly Glu LeuArg Gly His Val Ala Ala Leu Pro Tyr Cys 515 520 525 Gly His Ser Ala ArgHis Asp Thr Leu Pro Val Pro Leu Ala Gly 530 535 540 Ala Leu Val Leu ProPro Val Lys Ser Gln Ala Ala Gly His Ala 545 550 555 Trp Leu Ser Leu AspThr His Cys His Leu His Tyr Glu Val Leu 560 565 570 Leu Ala Gly Leu GlyGly Ser Glu Gln Gly Thr Val Thr Ala His 575 580 585 Leu Leu Gly Pro ProGly Thr Pro Gly Pro Arg Arg Leu Leu Lys 590 595 600 Gly Phe Tyr Gly SerGlu Ala Gln Gly Val Val Lys Asp Leu Glu 605 610 615 Pro Glu Leu Leu ArgHis Leu Ala Lys Gly Met Ala Ser Leu Met 620 625 630 Ile Thr Thr Lys GlySer Pro Arg Gly Glu Leu Arg Gly Gln Val 635 640 645 His Ile Ala Asn GlnCys Glu Val Gly Gly Leu Arg Leu Glu Ala 650 655 660 Ala Gly Ala Glu GlyVal Arg Ala Leu Gly Ala Pro Asp Thr Ala 665 670 675 Ser Ala Ala Pro ProVal Val Pro Gly Leu Pro Ala Leu Ala Pro 680 685 690 Ala Lys Pro Gly GlyPro Gly Arg Pro Arg Asp Pro Asn Thr Cys 695 700 705 Phe Phe Glu Gly GlnGln Arg Pro His Gly Ala Arg Trp Ala Pro 710 715 720 Asn Tyr Asp Pro LeuCys Ser Leu Cys Thr Cys Gln Arg Arg Thr 725 730 735 Val Ile Cys Asp ProVal Val Cys Pro Pro Pro Ser Cys Pro His 740 745 750 Pro Val Gln Ala ProAsp Gln Cys Cys Pro Val Cys Pro Glu Lys 755 760 765 Gln Asp Val Arg AspLeu Pro Gly Leu Pro Arg Ser Arg Asp Pro 770 775 780 Gly Glu Gly Cys TyrPhe Asp Gly Asp Arg Ser Trp Arg Ala Ala 785 790 795 Gly Thr Arg Trp HisPro Val Val Pro Pro Phe Gly Leu Ile Lys 800 805 810 Cys Ala Val Cys ThrCys Lys Gly Gly Thr Gly Glu Val His Cys 815 820 825 Glu Lys Val Gln CysPro Arg Leu Ala Cys Ala Gln Pro Val Arg 830 835 840 Val Asn Pro Thr AspCys Cys Lys Gln Cys Pro Val Gly Ser Gly 845 850 855 Ala His Pro Gln LeuGly Asp Pro Met Gln Ala Asp Gly Pro Arg 860 865 870 Gly Cys Arg Phe AlaGly Gln Trp Phe Pro Glu Ser Gln Ser Trp 875 880 885 His Pro Ser Val ProPro Phe Gly Glu Met Ser Cys Ile Thr Cys 890 895 900 Arg Cys Gly Ala GlyVal Pro His Cys Glu Arg Asp Asp Cys Ser 905 910 915 Leu Pro Leu Ser CysGly Ser Gly Lys Glu Ser Arg Cys Cys Ser 920 925 930 Arg Cys Thr Ala HisArg Arg Pro Pro Glu Thr Arg Thr Asp Pro 935 940 945 Glu Leu Glu Lys GluAla Glu Gly Ser 950 8 44 DNA Artificial Sequence SyntheticOligonucleotide probe 8 gactagttct agatcgcgag cggccgccct tttttttttt tttt44 9 28 DNA Artificial Sequence Synthetic oligonucleotide probe 9cggacgcgtg gggcctgcgc acccagct 28 10 36 DNA Artificial SequenceSynthetic oligonucleotide probe 10 gccgctcccc gaacgggcag cggctccttctcagaa 36 11 36 DNA Artificial Sequence Synthetic oligonucleotide probe11 ggcgcacagc acgcagcgca tcaccccgaa tggctc 36 12 26 DNA ArtificialSequence Synthetic Oligonucleotide Probe 12 gtgctgccca tccgttctga gaagga26 13 22 DNA Artificial Sequence Synthetic oligonucleotide probe 13gcagggtgct caaacaggac ac 22 14 3231 DNA Homo Sapien 14 ggcggagcagccctagccgc caccgtcgct ctcgcagctc tcgtcgccac 50 tgccaccgcc gccgccgtcactgcgtcctg gctccggctc ccgcgccctc 100 ccggccggcc atgcagcccc gccgcgcccaggcgcccggt gcgcagctgc 150 tgcccgcgct ggccctgctg ctgctgctgc tcggagcggggccccgaggc 200 agctccctgg ccaacccggt gcccgccgcg cccttgtctg cgcccgggcc250 gtgcgccgcg cagccctgcc ggaatggggg tgtgtgcacc tcgcgccctg 300agccggaccc gcagcacccg gcccccgccg gcgagcctgg ctacagctgc 350 acctgccccgccgggatctc cggcgccaac tgccagcttg ttgcagatcc 400 ttgtgccagc aacccttgtcaccatggcaa ctgcagcagc agcagcagca 450 gcagcagcga tggctacctc tgcatttgcaatgaaggcta tgaaggtccc 500 aactgtgaac aggcacttcc cagtctccca gccactggctggaccgaatc 550 catggcaccc cgacagcttc agcctgttcc tgctactcag gagcctgaca600 aaatcctgcc tcgctctcag gcaacggtga cactgcctac ctggcagccg 650aaaacagggc agaaagttgt agaaatgaaa tgggatcaag tggaggtgat 700 cccagatattgcctgtggga atgccagttc taacagctct gcgggtggcc 750 gcctggtatc ctttgaagtgccacagaaca cctcagtcaa gattcggcaa 800 gatgccactg cctcactgat tttgctctggaaggtcacgg ccacaggatt 850 ccaacagtgc tccctcatag atggacgaag tgtgaccccccttcaggctt 900 cagggggact ggtcctcctg gaggagatgc tcgccttggg gaataatcac950 tttattggtt ttgtgaatga ttctgtgact aagtctattg tggctttgcg 1000cttaactctg gtggtgaagg tcagcacctg tgtgccgggg gagagtcacg 1050 caaatgacttggagtgttca ggaaaaggaa aatgcaccac gaagccgtca 1100 gaggcaactt tttcctgtacctgtgaggag cagtacgtgg gtactttctg 1150 tgaagaatac gatgcttgcc agaggaaaccttgccaaaac aacgcgagct 1200 gtattgatgc aaatgaaaag caagatggga gcaatttcacctgtgtttgc 1250 cttcctggtt atactggaga gctttgccag tccaagattg attactgcat1300 cctagaccca tgcagaaatg gagcaacatg catttccagt ctcagtggat 1350tcacctgcca gtgtccagaa ggatacttcg gatctgcttg tgaagaaaag 1400 gtggacccctgcgcctcgtc tccgtgccag aacaacggca cctgctatgt 1450 ggacggggta cactttacctgcaactgcag cccgggcttc acagggccga 1500 cctgtgccca gcttattgac ttctgtgccctcagcccctg tgctcatggc 1550 acgtgccgca gcgtgggcac cagctacaaa tgcctctgtgatccaggtta 1600 ccatggcctc tactgtgagg aggaatataa tgagtgcctc tccgctccat1650 gcctgaatgc agccacctgc agggacctcg ttaatggcta tgagtgtgtg 1700tgcctggcag aatacaaagg aacacactgt gaattgtaca aggatccctg 1750 cgctaacgtcagctgtctga acggagccac ctgtgacagc gacggcctga 1800 atggcacgtg catctgtgcacccgggttta caggtgaaga gtgcgacatt 1850 gacataaatg aatgtgacag taacccctgccaccatggtg ggagctgcct 1900 ggaccagccc aatggttata actgccactg cccgcatggttgggtgggag 1950 caaactgtga gatccacctc caatggaagt ccgggcacat ggcggagagc2000 ctcaccaaca tgccacggca ctccctctac atcatcattg gagccctctg 2050cgtggccttc atccttatgc tgatcatcct gatcgtgggg atttgccgca 2100 tcagccgcattgaataccag ggttcttcca ggccagccta tgaggagttc 2150 tacaactgcc gcagcatcgacagcgagttc agcaatgcca ttgcatccat 2200 ccggcatgcc aggtttggaa agaaatcccggcctgcaatg tatgatgtga 2250 gccccatcgc ctatgaagat tacagtcctg atgacaaacccttggtcaca 2300 ctgattaaaa ctaaagattt gtaatctttt tttggattat ttttcaaaaa2350 gatgagatac tacactcatt taaatatttt taagaaaata aaaagcttaa 2400gaaatttaaa atgctagctg ctcaagagtt ttcagtagaa tatttaagaa 2450 ctaattttctgcagctttta gtttggaaaa aatattttaa aaacaaaatt 2500 tgtgaaacct atagacgatgttttaatgta ccttcagctc tctaaactgt 2550 gtgcttctac tagtgtgtgc tcttttcactgtagacacta tcacgagacc 2600 cagattaatt tctgtggttg ttacagaata agtctaatcaaggagaagtt 2650 tctgtttgac gtttgagtgc cggctttctg agtagagtta ggaaaaccac2700 gtaacgtagc atatgatgta taatagagta tacccgttac ttaaaaagaa 2750gtctgaaatg ttcgttttgt ggaaaagaaa ctagttaaat ttactattcc 2800 taacccgaatgaaattagcc tttgccttat tctgtgcatg ggtaagtaac 2850 ttatttctgc actgttttgttgaactttgt ggaaacattc tttcgagttt 2900 gtttttgtca ttttcgtaac agtcgtcgaactaggcctca aaaacatacg 2950 taacgaaaag gcctagcgag gcaaattctg attgatttgaatctatattt 3000 ttctttaaaa agtcaagggt tctatattgt gagtaaatta aatttacatt3050 tgagttgttt gttgctaaga ggtagtaaat gtaagagagt actggttcct 3100tcagtagtga gtatttctca tagtgcagct ttatttatct ccaggatgtt 3150 tttgtggctgtatttgattg atatgtgctt cttctgattc ttgctaattt 3200 ccaaccatat tgaataaatgtgatcaagtc a 3231 15 737 PRT Homo Sapien 15 Met Gln Pro Arg Arg Ala GlnAla Pro Gly Ala Gln Leu Leu Pro 1 5 10 15 Ala Leu Ala Leu Leu Leu LeuLeu Leu Gly Ala Gly Pro Arg Gly 20 25 30 Ser Ser Leu Ala Asn Pro Val ProAla Ala Pro Leu Ser Ala Pro 35 40 45 Gly Pro Cys Ala Ala Gln Pro Cys ArgAsn Gly Gly Val Cys Thr 50 55 60 Ser Arg Pro Glu Pro Asp Pro Gln His ProAla Pro Ala Gly Glu 65 70 75 Pro Gly Tyr Ser Cys Thr Cys Pro Ala Gly IleSer Gly Ala Asn 80 85 90 Cys Gln Leu Val Ala Asp Pro Cys Ala Ser Asn ProCys His His 95 100 105 Gly Asn Cys Ser Ser Ser Ser Ser Ser Ser Ser AspGly Tyr Leu 110 115 120 Cys Ile Cys Asn Glu Gly Tyr Glu Gly Pro Asn CysGlu Gln Ala 125 130 135 Leu Pro Ser Leu Pro Ala Thr Gly Trp Thr Glu SerMet Ala Pro 140 145 150 Arg Gln Leu Gln Pro Val Pro Ala Thr Gln Glu ProAsp Lys Ile 155 160 165 Leu Pro Arg Ser Gln Ala Thr Val Thr Leu Pro ThrTrp Gln Pro 170 175 180 Lys Thr Gly Gln Lys Val Val Glu Met Lys Trp AspGln Val Glu 185 190 195 Val Ile Pro Asp Ile Ala Cys Gly Asn Ala Ser SerAsn Ser Ser 200 205 210 Ala Gly Gly Arg Leu Val Ser Phe Glu Val Pro GlnAsn Thr Ser 215 220 225 Val Lys Ile Arg Gln Asp Ala Thr Ala Ser Leu IleLeu Leu Trp 230 235 240 Lys Val Thr Ala Thr Gly Phe Gln Gln Cys Ser LeuIle Asp Gly 245 250 255 Arg Ser Val Thr Pro Leu Gln Ala Ser Gly Gly LeuVal Leu Leu 260 265 270 Glu Glu Met Leu Ala Leu Gly Asn Asn His Phe IleGly Phe Val 275 280 285 Asn Asp Ser Val Thr Lys Ser Ile Val Ala Leu ArgLeu Thr Leu 290 295 300 Val Val Lys Val Ser Thr Cys Val Pro Gly Glu SerHis Ala Asn 305 310 315 Asp Leu Glu Cys Ser Gly Lys Gly Lys Cys Thr ThrLys Pro Ser 320 325 330 Glu Ala Thr Phe Ser Cys Thr Cys Glu Glu Gln TyrVal Gly Thr 335 340 345 Phe Cys Glu Glu Tyr Asp Ala Cys Gln Arg Lys ProCys Gln Asn 350 355 360 Asn Ala Ser Cys Ile Asp Ala Asn Glu Lys Gln AspGly Ser Asn 365 370 375 Phe Thr Cys Val Cys Leu Pro Gly Tyr Thr Gly GluLeu Cys Gln 380 385 390 Ser Lys Ile Asp Tyr Cys Ile Leu Asp Pro Cys ArgAsn Gly Ala 395 400 405 Thr Cys Ile Ser Ser Leu Ser Gly Phe Thr Cys GlnCys Pro Glu 410 415 420 Gly Tyr Phe Gly Ser Ala Cys Glu Glu Lys Val AspPro Cys Ala 425 430 435 Ser Ser Pro Cys Gln Asn Asn Gly Thr Cys Tyr ValAsp Gly Val 440 445 450 His Phe Thr Cys Asn Cys Ser Pro Gly Phe Thr GlyPro Thr Cys 455 460 465 Ala Gln Leu Ile Asp Phe Cys Ala Leu Ser Pro CysAla His Gly 470 475 480 Thr Cys Arg Ser Val Gly Thr Ser Tyr Lys Cys LeuCys Asp Pro 485 490 495 Gly Tyr His Gly Leu Tyr Cys Glu Glu Glu Tyr AsnGlu Cys Leu 500 505 510 Ser Ala Pro Cys Leu Asn Ala Ala Thr Cys Arg AspLeu Val Asn 515 520 525 Gly Tyr Glu Cys Val Cys Leu Ala Glu Tyr Lys GlyThr His Cys 530 535 540 Glu Leu Tyr Lys Asp Pro Cys Ala Asn Val Ser CysLeu Asn Gly 545 550 555 Ala Thr Cys Asp Ser Asp Gly Leu Asn Gly Thr CysIle Cys Ala 560 565 570 Pro Gly Phe Thr Gly Glu Glu Cys Asp Ile Asp IleAsn Glu Cys 575 580 585 Asp Ser Asn Pro Cys His His Gly Gly Ser Cys LeuAsp Gln Pro 590 595 600 Asn Gly Tyr Asn Cys His Cys Pro His Gly Trp ValGly Ala Asn 605 610 615 Cys Glu Ile His Leu Gln Trp Lys Ser Gly His MetAla Glu Ser 620 625 630 Leu Thr Asn Met Pro Arg His Ser Leu Tyr Ile IleIle Gly Ala 635 640 645 Leu Cys Val Ala Phe Ile Leu Met Leu Ile Ile LeuIle Val Gly 650 655 660 Ile Cys Arg Ile Ser Arg Ile Glu Tyr Gln Gly SerSer Arg Pro 665 670 675 Ala Tyr Glu Glu Phe Tyr Asn Cys Arg Ser Ile AspSer Glu Phe 680 685 690 Ser Asn Ala Ile Ala Ser Ile Arg His Ala Arg PheGly Lys Lys 695 700 705 Ser Arg Pro Ala Met Tyr Asp Val Ser Pro Ile AlaTyr Glu Asp 710 715 720 Tyr Ser Pro Asp Asp Lys Pro Leu Val Thr Leu IleLys Thr Lys 725 730 735 Asp Leu 16 43 DNA Artificial Sequence SyntheticOligonucleotide Probe 16 tgtaaaacga cggccagtta aatagacctg caattattaa tct43 17 41 DNA Artificial Sequence Synthetic Oligonucleotide Probe 17caggaaacag ctatgaccac ctgcacacct gcaaatccat t 41 18 508 DNA Homo Sapien18 ctctggaagg tcacggccac aggattccaa cagtgctccc tcatagatgg 50 acgaaagtgtgacccccctt tcaggctttc agggggactg gtcctcctgg 100 aggagatgct cgccttggggaataatcact ttattggttt tgtgaatgat 150 tctgtgacta agtctattgt ggctttgcgcttaactctgg tggtgaaggt 200 cagcacctgt gtgccggggg agagtcacgc aaatgacttggagtgttcag 250 gaaaaggaaa atgcaccacg aagccgtcag aggcaacttt ttcctgtacc300 tgtgaggagc agtacgtggg tactttctgt gaagaatacg atgcttgcca 350gaggaaacct tgccaaaaca acgcgagctg tattgatgca aatgaaaagc 400 aagatgggagcaatttcacc tgtgtttgcc ttcctggtta tactggagag 450 ctttgccaac cgaactgagattggagcgaa cgacctacac cgaactgaga 500 taggggag 508 19 508 DNA Homo Sapien19 ctctggaagg tcacggccac aggattccaa cagtgctccc tcatagatgg 50 acgaaagtgtgacccccctt tcaggctttc agggggactg gtcctcctgg 100 aggagatgct cgccttggggaataatcact ttattggttt tgtgaatgat 150 tctgtgacta agtctattgt ggctttgcgcttaactctgg tggtgaaggt 200 cagcacctgt gtgccggggg agagtcacgc aaatgacttggagtgttcag 250 gaaaaggaaa atgcaccacg aagccgtcag aggcaacttt ttcctgtacc300 tgtgaggagc agtacgtggg tactttctgt gaagaatacg atgcttgcca 350gaggaaacct tgccaaaaca acgcgagctg tattgatgca aatgaaaagc 400 aagatgggagcaatttcacc tgtgtttgcc ttcctggtta tactggagag 450 ctttgccaac cgaactgagattggagcgaa cgacctacac cgaactgaga 500 taggggag 508 20 23 DNA ArtificialSequence Synthetic Oligonucleotide Probe 20 ctctggaagg tcacggccac agg 2321 24 DNA Artificial Sequence Synthetic oligonucleotide probe 21ctcagttcgg ttggcaaagc tctc 24 22 69 DNA Artificial Sequence Syntheticoligonucleotide probe 22 cagtgctccc tcatagatgg acgaaagtgt gacccccctttcaggcgaga 50 gctttgccaa ccgaactga 69 23 1520 DNA Homo Sapien 23gctgagtctg ctgctcctgc tgctgctgct ccagcctgta acctgtgcct 50 acaccacgccaggccccccc agagccctca ccacgctggg cgcccccaga 100 gcccacacca tgccgggcacctacgctccc tcgaccacac tcagtagtcc 150 cagcacccag ggcctgcaag agcaggcacgggccctgatg cgggacttcc 200 cgctcgtgga cggccacaac gacctgcccc tggtcctaaggcaggtttac 250 cagaaagggc tacaggatgt taacctgcgc aatttcagct acggccagac300 cagcctggac aggcttagag atggcctcgt gggcgcccag ttctggtcag 350cctatgtgcc atgccagacc caggaccggg atgccctgcg cctcaccctg 400 gagcagattgacctcatacg ccgcatgtgt gcctcctatt ctgagctgga 450 gcttgtgacc tcggctaaagctctgaacga cactcagaaa ttggcctgcc 500 tcatcggtgt agagggtggc cactcgctggacaatagcct ctccatctta 550 cgtaccttct acatgctggg agtgcgctac ctgacgctcacccacacctg 600 caacacaccc tgggcagaga gctccgctaa gggcgtccac tccttctaca650 acaacatcag cgggctgact gactttggtg agaaggtggt ggcagaaatg 700aaccgcctgg gcatgatggt agacttatcc catgtctcag atgctgtggc 750 acggcgggccctggaagtgt cacaggcacc tgtgatcttc tcccactcgg 800 ctgcccgggg tgtgtgcaacagtgctcgga atgttcctga tgacatcctg 850 cagcttctga agaagaacgg tggcgtcgtgatggtgtctt tgtccatggg 900 agtaatacag tgcaacccat cagccaatgt gtccactgtggcagatcact 950 tcgaccacat caaggctgtc attggatcca agttcatcgg gattggtgga1000 gattatgatg gggccggcaa attccctcag gggctggaag acgtgtccac 1050atacccggtc ctgatagagg agttgctgag tcgtggctgg agtgaggaag 1100 agcttcagggtgtccttcgt ggaaacctgc tgcgggtctt cagacaagtg 1150 gaaaaggtac aggaagaaaacaaatggcaa agccccttgg aggacaagtt 1200 cccggatgag cagctgagca gttcctgccactccgacctc tcacgtctgc 1250 gtcagagaca gagtctgact tcaggccagg aactcactgagattcccata 1300 cactggacag ccaagttacc agccaagtgg tcagtctcag agtcctcccc1350 ccacatggcc ccagtccttg cagttgtggc caccttccca gtccttattc 1400tgtggctctg atgacccagt tagtcctgcc agatgtcact gtagcaagcc 1450 acagacaccccacaaagttc ccctgttgtg caggcacaaa tatttcctga 1500 aataaatgtt ttggacatag1520 24 433 PRT Homo Sapien 24 Met Pro Gly Thr Tyr Ala Pro Ser Thr ThrLeu Ser Ser Pro Ser 1 5 10 15 Thr Gln Gly Leu Gln Glu Gln Ala Arg AlaLeu Met Arg Asp Phe 20 25 30 Pro Leu Val Asp Gly His Asn Asp Leu Pro LeuVal Leu Arg Gln 35 40 45 Val Tyr Gln Lys Gly Leu Gln Asp Val Asn Leu ArgAsn Phe Ser 50 55 60 Tyr Gly Gln Thr Ser Leu Asp Arg Leu Arg Asp Gly LeuVal Gly 65 70 75 Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gln Thr Gln AspArg 80 85 90 Asp Ala Leu Arg Leu Thr Leu Glu Gln Ile Asp Leu Ile Arg Arg95 100 105 Met Cys Ala Ser Tyr Ser Glu Leu Glu Leu Val Thr Ser Ala Lys110 115 120 Ala Leu Asn Asp Thr Gln Lys Leu Ala Cys Leu Ile Gly Val Glu125 130 135 Gly Gly His Ser Leu Asp Asn Ser Leu Ser Ile Leu Arg Thr Phe140 145 150 Tyr Met Leu Gly Val Arg Tyr Leu Thr Leu Thr His Thr Cys Asn155 160 165 Thr Pro Trp Ala Glu Ser Ser Ala Lys Gly Val His Ser Phe Tyr170 175 180 Asn Asn Ile Ser Gly Leu Thr Asp Phe Gly Glu Lys Val Val Ala185 190 195 Glu Met Asn Arg Leu Gly Met Met Val Asp Leu Ser His Val Ser200 205 210 Asp Ala Val Ala Arg Arg Ala Leu Glu Val Ser Gln Ala Pro Val215 220 225 Ile Phe Ser His Ser Ala Ala Arg Gly Val Cys Asn Ser Ala Arg230 235 240 Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Lys Lys Asn Gly Gly245 250 255 Val Val Met Val Ser Leu Ser Met Gly Val Ile Gln Cys Asn Pro260 265 270 Ser Ala Asn Val Ser Thr Val Ala Asp His Phe Asp His Ile Lys275 280 285 Ala Val Ile Gly Ser Lys Phe Ile Gly Ile Gly Gly Asp Tyr Asp290 295 300 Gly Ala Gly Lys Phe Pro Gln Gly Leu Glu Asp Val Ser Thr Tyr305 310 315 Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gly Trp Ser Glu Glu320 325 330 Glu Leu Gln Gly Val Leu Arg Gly Asn Leu Leu Arg Val Phe Arg335 340 345 Gln Val Glu Lys Val Gln Glu Glu Asn Lys Trp Gln Ser Pro Leu350 355 360 Glu Asp Lys Phe Pro Asp Glu Gln Leu Ser Ser Ser Cys His Ser365 370 375 Asp Leu Ser Arg Leu Arg Gln Arg Gln Ser Leu Thr Ser Gly Gln380 385 390 Glu Leu Thr Glu Ile Pro Ile His Trp Thr Ala Lys Leu Pro Ala395 400 405 Lys Trp Ser Val Ser Glu Ser Ser Pro His Met Ala Pro Val Leu410 415 420 Ala Val Val Ala Thr Phe Pro Val Leu Ile Leu Trp Leu 425 43025 22 DNA Artificial Sequence Synthetic oligonucleotide probe 25agttctggtc agcctatgtg cc 22 26 24 DNA Artificial Sequence Syntheticoligonucleotide probe 26 cgtgatggtg tctttgtcca tggg 24 27 24 DNAArtificial Sequence Synthetic oligonucleotide probe 27 ctccaccaatcccgatgaac ttgg 24 28 50 DNA Artificial Sequence Syntheticoligonucleotide probe 28 gagcagattg acctcatacg ccgcatgtgt gcctcctattctgagctgga 50 29 1416 DNA Homo Sapien 29 aaaacctata aatattccggattattcata ccgtcccacc atcgggcgcg 50 gatccgcggc cgcgaattct aaaccaacatgccgggcacc tacgctccct 100 cgaccacact cagtagtccc agcacccagg gcctgcaagagcaggcacgg 150 gccctgatgc gggacttccc gctcgtggac ggccacaacg acctgcccct200 ggtcctaagg caggtttacc agaaagggct acaggatgtt aacctgcgca 250atttcagcta cggccagacc agcctggaca ggcttagaga tggcctcgtg 300 ggcgcccagttctggtcagc ctatgtgcca tgccagaccc aggaccggga 350 tgccctgcgc ctcaccctggagcagattga cctcatacgc cgcatgtgtg 400 cctcctattc tgagctggag cttgtgacctcggctaaagc tctgaacgac 450 actcagaaat tggcctgcct catcggtgta gagggtggccactcgctgga 500 caatagcctc tccatcttac gtaccttcta catgctggga gtgcgctacc550 tgacgctcac ccacacctgc aacacaccct gggcagagag ctccgctaag 600ggcgtccact ccttctacaa caacatcagc gggctgactg actttggtga 650 gaaggtggtggcagaaatga accgcctggg catgatggta gacttatccc 700 atgtctcaga tgctgtggcacggcgggccc tggaagtgtc acaggcacct 750 gtgatcttct cccactcggc tgcccggggtgtgtgcaaca gtgctcggaa 800 tgttcctgat gacatcctgc agcttctgaa gaagaacggtggcgtcgtga 850 tggtgtcttt gtccatggga gtaatacagt gcaacccatc agccaatgtg900 tccactgtgg cagatcactt cgaccacatc aaggctgtca ttggatccaa 950gttcatcggg attggtggag attatgatgg ggccggcaaa ttccctcagg 1000 ggctggaagacgtgtccaca tacccggtcc tgatagagga gttgctgagt 1050 cgtggctgga gtgaggaagagcttcagggt gtccttcgtg gaaacctgct 1100 gcgggtcttc agacaagtgg aaaaggtacaggaagaaaac aaatggcaaa 1150 gccccttgga ggacaagttc ccggatgagc agctgagcagttcctgccac 1200 tccgacctct cacgtctgcg tcagagacag agtctgactt caggccagga1250 actcactgag attcccatac actggacagc caagttacca gccaagtggt 1300cagtctcaga gtcctccccc caccctgaca aaactcacac atgcccaccg 1350 tgcccagcacctgaactcct ggggggaccg tcagtcttcc tcttcccccc 1400 aaaacccaag gacacc 141630 446 PRT Homo Sapien 30 Met Pro Gly Thr Tyr Ala Pro Ser Thr Thr LeuSer Ser Pro Ser 1 5 10 15 Thr Gln Gly Leu Gln Glu Gln Ala Arg Ala LeuMet Arg Asp Phe 20 25 30 Pro Leu Val Asp Gly His Asn Asp Leu Pro Leu ValLeu Arg Gln 35 40 45 Val Tyr Gln Lys Gly Leu Gln Asp Val Asn Leu Arg AsnPhe Ser 50 55 60 Tyr Gly Gln Thr Ser Leu Asp Arg Leu Arg Asp Gly Leu ValGly 65 70 75 Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gln Thr Gln Asp Arg80 85 90 Asp Ala Leu Arg Leu Thr Leu Glu Gln Ile Asp Leu Ile Arg Arg 95100 105 Met Cys Ala Ser Tyr Ser Glu Leu Glu Leu Val Thr Ser Ala Lys 110115 120 Ala Leu Asn Asp Thr Gln Lys Leu Ala Cys Leu Ile Gly Val Glu 125130 135 Gly Gly His Ser Leu Asp Asn Ser Leu Ser Ile Leu Arg Thr Phe 140145 150 Tyr Met Leu Gly Val Arg Tyr Leu Thr Leu Thr His Thr Cys Asn 155160 165 Thr Pro Trp Ala Glu Ser Ser Ala Lys Gly Val His Ser Phe Tyr 170175 180 Asn Asn Ile Ser Gly Leu Thr Asp Phe Gly Glu Lys Val Val Ala 185190 195 Glu Met Asn Arg Leu Gly Met Met Val Asp Leu Ser His Val Ser 200205 210 Asp Ala Val Ala Arg Arg Ala Leu Glu Val Ser Gln Ala Pro Val 215220 225 Ile Phe Ser His Ser Ala Ala Arg Gly Val Cys Asn Ser Ala Arg 230235 240 Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Lys Lys Asn Gly Gly 245250 255 Val Val Met Val Ser Leu Ser Met Gly Val Ile Gln Cys Asn Pro 260265 270 Ser Ala Asn Val Ser Thr Val Ala Asp His Phe Asp His Ile Lys 275280 285 Ala Val Ile Gly Ser Lys Phe Ile Gly Ile Gly Gly Asp Tyr Asp 290295 300 Gly Ala Gly Lys Phe Pro Gln Gly Leu Glu Asp Val Ser Thr Tyr 305310 315 Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gly Trp Ser Glu Glu 320325 330 Glu Leu Gln Gly Val Leu Arg Gly Asn Leu Leu Arg Val Phe Arg 335340 345 Gln Val Glu Lys Val Gln Glu Glu Asn Lys Trp Gln Ser Pro Leu 350355 360 Glu Asp Lys Phe Pro Asp Glu Gln Leu Ser Ser Ser Cys His Ser 365370 375 Asp Leu Ser Arg Leu Arg Gln Arg Gln Ser Leu Thr Ser Gly Gln 380385 390 Glu Leu Thr Glu Ile Pro Ile His Trp Thr Ala Lys Leu Pro Ala 395400 405 Lys Trp Ser Val Ser Glu Ser Ser Pro His Pro Asp Lys Thr His 410415 420 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 425430 435 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 440 445 31 1790 DNAHomo Sapien 31 cgcccagcga cgtgcgggcg gcctggcccg cgccctcccg cgcccggcct 50gcgtcccgcg ccctgcgcca ccgccgccga gccgcagccc gccgcgcgcc 100 cccggcagcgccggccccat gcccgccggc cgccggggcc ccgccgccca 150 atccgcgcgg cggccgccgccgttgctgcc cctgctgctg ctgctctgcg 200 tcctcggggc gccgcgagcc ggatcaggagcccacacagc tgtgatcagt 250 ccccaggatc ccacgcttct catcggctcc tccctgctggccacctgctc 300 agtgcacgga gacccaccag gagccaccgc cgagggcctc tactggaccc350 tcaacgggcg ccgcctgccc cctgagctct cccgtgtact caacgcctcc 400accttggctc tggccctggc caacctcaat gggtccaggc agcggtcggg 450 ggacaacctcgtgtgccacg cccgtgacgg cagcatcctg gctggctcct 500 gcctctatgt tggcctgcccccagagaaac ccgtcaacat cagctgctgg 550 tccaagaaca tgaaggactt gacctgccgctggacgccag gggcccacgg 600 ggagaccttc ctccacacca actactccct caagtacaagcttaggtggt 650 atggccagga caacacatgt gaggagtacc acacagtggg gccccactcc700 tgccacatcc ccaaggacct ggctctcttt acgccctatg agatctgggt 750ggaggccacc aaccgcctgg gctctgcccg ctccgatgta ctcacgctgg 800 atatcctggatgtggtgacc acggaccccc cgcccgacgt gcacgtgagc 850 cgcgtcgggg gcctggaggaccagctgagc gtgcgctggg tgtcgccacc 900 cgccctcaag gatttcctct ttcaagccaaataccagatc cgctaccgag 950 tggaggacag tgtggactgg aaggtggtgg acgatgtgagcaaccagacc 1000 tcctgccgcc tggccggcct gaaacccggc accgtgtact tcgtgcaagt1050 gcgctgcaac ccctttggca tctatggctc caagaaagcc gggatctgga 1100gtgagtggag ccaccccaca gccgcctcca ctccccgcag tgagcgcccg 1150 ggcccgggcggcggggcgtg cgaaccgcgg ggcggagagc cgagctcggg 1200 gccggtgcgg cgcgagctcaagcagttcct gggctggctc aagaagcacg 1250 cgtactgctc caacctcagc ttccgcctctacgaccagtg gcgagcctgg 1300 atgcagaagt cgcacaagac ccgcaaccag gacgaggggatcctgccctc 1350 gggcagacgg ggcacggcga gaggtcctgc cagataagct gtaggggctc1400 aggccaccct ccctgccacg tggagacgca gaggccgaac ccaaactggg 1450gccacctctg taccctcact tcagggcacc tgagccaccc tcagcaggag 1500 ctggggtggcccctgagctc caacggccat aacagctctg actcccacgt 1550 gaggccacct ttgggtgcaccccagtgggt gtgtgtgtgt gtgtgagggt 1600 tggttgagtt gcctagaacc cctgccagggctgggggtga gaaggggagt 1650 cattactccc cattacctag ggcccctcca aaagagtccttttaaataaa 1700 tgagctattt aggtgctgtg attgtgaaaa aaaaaaaaaa aaaaaaaaaa1750 aaaaaaaaaa aaaaaaaaaa aaaaacaaaa aaaaaaaaaa 1790 32 422 PRT HomoSapien 32 Met Pro Ala Gly Arg Arg Gly Pro Ala Ala Gln Ser Ala Arg Arg 15 10 15 Pro Pro Pro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly 2025 30 Ala Pro Arg Ala Gly Ser Gly Ala His Thr Ala Val Ile Ser Pro 35 4045 Gln Asp Pro Thr Leu Leu Ile Gly Ser Ser Leu Leu Ala Thr Cys 50 55 60Ser Val His Gly Asp Pro Pro Gly Ala Thr Ala Glu Gly Leu Tyr 65 70 75 TrpThr Leu Asn Gly Arg Arg Leu Pro Pro Glu Leu Ser Arg Val 80 85 90 Leu AsnAla Ser Thr Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly 95 100 105 Ser ArgGln Arg Ser Gly Asp Asn Leu Val Cys His Ala Arg Asp 110 115 120 Gly SerIle Leu Ala Gly Ser Cys Leu Tyr Val Gly Leu Pro Pro 125 130 135 Glu LysPro Val Asn Ile Ser Cys Trp Ser Lys Asn Met Lys Asp 140 145 150 Leu ThrCys Arg Trp Thr Pro Gly Ala His Gly Glu Thr Phe Leu 155 160 165 His ThrAsn Tyr Ser Leu Lys Tyr Lys Leu Arg Trp Tyr Gly Gln 170 175 180 Asp AsnThr Cys Glu Glu Tyr His Thr Val Gly Pro His Ser Cys 185 190 195 His IlePro Lys Asp Leu Ala Leu Phe Thr Pro Tyr Glu Ile Trp 200 205 210 Val GluAla Thr Asn Arg Leu Gly Ser Ala Arg Ser Asp Val Leu 215 220 225 Thr LeuAsp Ile Leu Asp Val Val Thr Thr Asp Pro Pro Pro Asp 230 235 240 Val HisVal Ser Arg Val Gly Gly Leu Glu Asp Gln Leu Ser Val 245 250 255 Arg TrpVal Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gln Ala 260 265 270 Lys TyrGln Ile Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys 275 280 285 Val ValAsp Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Ala Gly 290 295 300 Leu LysPro Gly Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro 305 310 315 Phe GlyIle Tyr Gly Ser Lys Lys Ala Gly Ile Trp Ser Glu Trp 320 325 330 Ser HisPro Thr Ala Ala Ser Thr Pro Arg Ser Glu Arg Pro Gly 335 340 345 Pro GlyGly Gly Ala Cys Glu Pro Arg Gly Gly Glu Pro Ser Ser 350 355 360 Gly ProVal Arg Arg Glu Leu Lys Gln Phe Leu Gly Trp Leu Lys 365 370 375 Lys HisAla Tyr Cys Ser Asn Leu Ser Phe Arg Leu Tyr Asp Gln 380 385 390 Trp ArgAla Trp Met Gln Lys Ser His Lys Thr Arg Asn Gln Asp 395 400 405 Glu GlyIle Leu Pro Ser Gly Arg Arg Gly Thr Ala Arg Gly Pro 410 415 420 Ala Arg33 23 DNA Artificial Sequence Synthetic oligonucleotide probe 33cccgcccgac gtgcacgtga gcc 23 34 23 DNA Artificial Sequence Syntheticoligonucleotide probe 34 tgagccagcc caggaactgc ttg 23 35 50 DNAArtificial Sequence Synthetic oligonucleotide probe 35 caagtgcgctgcaacccctt tggcatctat ggctccaaga aagccgggat 50 36 1771 DNA Homo Sapien36 cccacgcgtc cgctggtgtt agatcgagca accctctaaa agcagtttag 50 agtggtaaaaaaaaaaaaaa acacaccaaa cgctcgcagc cacaaaaggg 100 atgaaatttc ttctggacatcctcctgctt ctcccgttac tgatcgtctg 150 ctccctagag tccttcgtga agctttttattcctaagagg agaaaatcag 200 tcaccggcga aatcgtgctg attacaggag ctgggcatggaattgggaga 250 ctgactgcct atgaatttgc taaacttaaa agcaagctgg ttctctggga300 tataaataag catggactgg aggaaacagc tgccaaatgc aagggactgg 350gtgccaaggt tcataccttt gtggtagact gcagcaaccg agaagatatt 400 tacagctctgcaaagaaggt gaaggcagaa attggagatg ttagtatttt 450 agtaaataat gctggtgtagtctatacatc agatttgttt gctacacaag 500 atcctcagat tgaaaagact tttgaagttaatgtacttgc acatttctgg 550 actacaaagg catttcttcc tgcaatgacg aagaataaccatggccatat 600 tgtcactgtg gcttcggcag ctggacatgt ctcggtcccc ttcttactgg650 cttactgttc aagcaagttt gctgctgttg gatttcataa aactttgaca 700gatgaactgg ctgccttaca aataactgga gtcaaaacaa catgtctgtg 750 tcctaatttcgtaaacactg gcttcatcaa aaatccaagt acaagtttgg 800 gacccactct ggaacctgaggaagtggtaa acaggctgat gcatgggatt 850 ctgactgagc agaagatgat ttttattccatcttctatag cttttttaac 900 aacattggaa aggatccttc ctgagcgttt cctggcagttttaaaacgaa 950 aaatcagtgt taagtttgat gcagttattg gatataaaat gaaagcgcaa1000 taagcaccta gttttctgaa aactgattta ccaggtttag gttgatgtca 1050tctaatagtg ccagaatttt aatgtttgaa cttctgtttt ttctaattat 1100 ccccatttcttcaatatcat ttttgaggct ttggcagtct tcatttacta 1150 ccacttgttc tttagccaaaagctgattac atatgatata aacagagaaa 1200 tacctttaga ggtgacttta aggaaaatgaagaaaaagaa ccaaaatgac 1250 tttattaaaa taatttccaa gattatttgt ggctcacctgaaggctttgc 1300 aaaatttgta ccataaccgt ttatttaaca tatattttta tttttgattg1350 cacttaaatt ttgtataatt tgtgtttctt tttctgttct acataaaatc 1400agaaacttca agctctctaa ataaaatgaa ggactatatc tagtggtatt 1450 tcacaatgaatatcatgaac tctcaatggg taggtttcat cctacccatt 1500 gccactctgt ttcctgagagatacctcaca ttccaatgcc aaacatttct 1550 gcacagggaa gctagaggtg gatacacgtgttgcaagtat aaaagcatca 1600 ctgggattta aggagaattg agagaatgta cccacaaatggcagcaataa 1650 taaatggatc acacttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa1700 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1750aaaaaaaaaa aaaaaaaaaa a 1771 37 300 PRT Homo Sapien 37 Met Lys Phe LeuLeu Asp Ile Leu Leu Leu Leu Pro Leu Leu Ile 1 5 10 15 Val Cys Ser LeuGlu Ser Phe Val Lys Leu Phe Ile Pro Lys Arg 20 25 30 Arg Lys Ser Val ThrGly Glu Ile Val Leu Ile Thr Gly Ala Gly 35 40 45 His Gly Ile Gly Arg LeuThr Ala Tyr Glu Phe Ala Lys Leu Lys 50 55 60 Ser Lys Leu Val Leu Trp AspIle Asn Lys His Gly Leu Glu Glu 65 70 75 Thr Ala Ala Lys Cys Lys Gly LeuGly Ala Lys Val His Thr Phe 80 85 90 Val Val Asp Cys Ser Asn Arg Glu AspIle Tyr Ser Ser Ala Lys 95 100 105 Lys Val Lys Ala Glu Ile Gly Asp ValSer Ile Leu Val Asn Asn 110 115 120 Ala Gly Val Val Tyr Thr Ser Asp LeuPhe Ala Thr Gln Asp Pro 125 130 135 Gln Ile Glu Lys Thr Phe Glu Val AsnVal Leu Ala His Phe Trp 140 145 150 Thr Thr Lys Ala Phe Leu Pro Ala MetThr Lys Asn Asn His Gly 155 160 165 His Ile Val Thr Val Ala Ser Ala AlaGly His Val Ser Val Pro 170 175 180 Phe Leu Leu Ala Tyr Cys Ser Ser LysPhe Ala Ala Val Gly Phe 185 190 195 His Lys Thr Leu Thr Asp Glu Leu AlaAla Leu Gln Ile Thr Gly 200 205 210 Val Lys Thr Thr Cys Leu Cys Pro AsnPhe Val Asn Thr Gly Phe 215 220 225 Ile Lys Asn Pro Ser Thr Ser Leu GlyPro Thr Leu Glu Pro Glu 230 235 240 Glu Val Val Asn Arg Leu Met His GlyIle Leu Thr Glu Gln Lys 245 250 255 Met Ile Phe Ile Pro Ser Ser Ile AlaPhe Leu Thr Thr Leu Glu 260 265 270 Arg Ile Leu Pro Glu Arg Phe Leu AlaVal Leu Lys Arg Lys Ile 275 280 285 Ser Val Lys Phe Asp Ala Val Ile GlyTyr Lys Met Lys Ala Gln 290 295 300 38 23 DNA Artificial SequenceSynthetic oligonucleotide probe 38 ggtgaaggca gaaattggag atg 23 39 24DNA Artificial Sequence Synthetic oligonucleotide probe 39 atcccatgcatcagcctgtt tacc 24 40 48 DNA Artificial Sequence Syntheticoligonucleotide probe 40 gctggtgtag tctatacatc agatttgttt gctacacaagatcctcag 48 41 1377 DNA Homo Sapien 41 gactagttct cttggagtct gggaggaggaaagcggagcc ggcagggagc 50 gaaccaggac tggggtgacg gcagggcagg gggcgcctggccggggagaa 100 gcgcgggggc tggagcacca ccaactggag ggtccggagt agcgagcgcc150 ccgaaggagg ccatcgggga gccgggaggg gggactgcga gaggaccccg 200gcgtccgggc tcccggtgcc agcgctatga ggccactcct cgtcctgctg 250 ctcctgggcctggcggccgg ctcgccccca ctggacgaca acaagatccc 300 cagcctctgc ccggggcaccccggccttcc aggcacgccg ggccaccatg 350 gcagccaggg cttgccgggc cgcgatggccgcgacggccg cgacggcgcg 400 cccggggctc cgggagagaa aggcgagggc gggaggccgggactgccggg 450 acctcgaggg gaccccgggc cgcgaggaga ggcgggaccc gcggggccca500 ccgggcctgc cggggagtgc tcggtgcctc cgcgatccgc cttcagcgcc 550aagcgctccg agagccgggt gcctccgccg tctgacgcac ccttgccctt 600 cgaccgcgtgctggtgaacg agcagggaca ttacgacgcc gtcaccggca 650 agttcacctg ccaggtgcctggggtctact acttcgccgt ccatgccacc 700 gtctaccggg ccagcctgca gtttgatctggtgaagaatg gcgaatccat 750 tgcctctttc ttccagtttt tcggggggtg gcccaagccagcctcgctct 800 cggggggggc catggtgagg ctggagcctg aggaccaagt gtgggtgcag850 gtgggtgtgg gtgactacat tggcatctat gccagcatca agacagacag 900caccttctcc ggatttctgg tgtactccga ctggcacagc tccccagtct 950 ttgcttagtgcccactgcaa agtgagctca tgctctcact cctagaagga 1000 gggtgtgagg ctgacaaccaggtcatccag gagggctggc ccccctggaa 1050 tattgtgaat gactagggag gtggggtagagcactctccg tcctgctgct 1100 ggcaaggaat gggaacagtg gctgtctgcg atcaggtctggcagcatggg 1150 gcagtggctg gatttctgcc caagaccaga ggagtgtgct gtgctggcaa1200 gtgtaagtcc cccagttgct ctggtccagg agcccacggt ggggtgctct 1250cttcctggtc ctctgcttct ctggatcctc cccaccccct cctgctcctg 1300 gggccggcccttttctcaga gatcactcaa taaacctaag aaccctcata 1350 aaaaaaaaaa aaaaaaaaaaaaaaaaa 1377 42 243 PRT Homo Sapien 42 Met Arg Pro Leu Leu Val Leu LeuLeu Leu Gly Leu Ala Ala Gly 1 5 10 15 Ser Pro Pro Leu Asp Asp Asn LysIle Pro Ser Leu Cys Pro Gly 20 25 30 His Pro Gly Leu Pro Gly Thr Pro GlyHis His Gly Ser Gln Gly 35 40 45 Leu Pro Gly Arg Asp Gly Arg Asp Gly ArgAsp Gly Ala Pro Gly 50 55 60 Ala Pro Gly Glu Lys Gly Glu Gly Gly Arg ProGly Leu Pro Gly 65 70 75 Pro Arg Gly Asp Pro Gly Pro Arg Gly Glu Ala GlyPro Ala Gly 80 85 90 Pro Thr Gly Pro Ala Gly Glu Cys Ser Val Pro Pro ArgSer Ala 95 100 105 Phe Ser Ala Lys Arg Ser Glu Ser Arg Val Pro Pro ProSer Asp 110 115 120 Ala Pro Leu Pro Phe Asp Arg Val Leu Val Asn Glu GlnGly His 125 130 135 Tyr Asp Ala Val Thr Gly Lys Phe Thr Cys Gln Val ProGly Val 140 145 150 Tyr Tyr Phe Ala Val His Ala Thr Val Tyr Arg Ala SerLeu Gln 155 160 165 Phe Asp Leu Val Lys Asn Gly Glu Ser Ile Ala Ser PhePhe Gln 170 175 180 Phe Phe Gly Gly Trp Pro Lys Pro Ala Ser Leu Ser GlyGly Ala 185 190 195 Met Val Arg Leu Glu Pro Glu Asp Gln Val Trp Val GlnVal Gly 200 205 210 Val Gly Asp Tyr Ile Gly Ile Tyr Ala Ser Ile Lys ThrAsp Ser 215 220 225 Thr Phe Ser Gly Phe Leu Val Tyr Ser Asp Trp His SerSer Pro 230 235 240 Val Phe Ala 43 24 DNA Artificial Sequence Syntheticoligonucleotide probe 43 tacaggccca gtcaggacca gggg 24 44 18 DNAArtificial Sequence Synthetic oligonucleotide probe 44 agccagcctcgctctcgg 18 45 18 DNA Artificial Sequence Synthetic oligonucleotideprobe 45 gtctgcgatc aggtctgg 18 46 20 DNA Artificial Sequence Syntheticoligonucleotide probe 46 gaaagaggca atggattcgc 20 47 24 DNA ArtificialSequence Synthetic oligonucleotide probe 47 gacttacact tgccagcaca gcac24 48 45 DNA Artificial Sequence Synthetic oligonucleotide probe 48ggagcaccac caactggagg gtccggagta gcgagcgccc cgaag 45 49 1876 DNA HomoSapien 49 ctcttttgtc caccagccca gcctgactcc tggagattgt gaatagctcc 50atccagcctg agaaacaagc cgggtggctg agccaggctg tgcacggagc 100 acctgacgggcccaacagac ccatgctgca tccagagacc tcccctggcc 150 gggggcatct cctggctgtgctcctggccc tccttggcac cacctgggca 200 gaggtgtggc caccccagct gcaggagcaggctccgatgg ccggagccct 250 gaacaggaag gagagtttct tgctcctctc cctgcacaaccgcctgcgca 300 gctgggtcca gccccctgcg gctgacatgc ggaggctgga ctggagtgac350 agcctggccc aactggctca agccagggca gccctctgtg gaatcccaac 400cccgagcctg gcatccggcc tgtggcgcac cctgcaagtg ggctggaaca 450 tgcagctgctgcccgcgggc ttggcgtcct ttgttgaagt ggtcagccta 500 tggtttgcag aggggcagcggtacagccac gcggcaggag agtgtgctcg 550 caacgccacc tgcacccact acacgcagctcgtgtgggcc acctcaagcc 600 agctgggctg tgggcggcac ctgtgctctg caggccagacagcgatagaa 650 gcctttgtct gtgcctactc ccccggaggc aactgggagg tcaacgggaa700 gacaatcatc ccctataaga agggtgcctg gtgttcgctc tgcacagcca 750gtgtctcagg ctgcttcaaa gcctgggacc atgcaggggg gctctgtgag 800 gtccccaggaatccttgtcg catgagctgc cagaaccatg gacgtctcaa 850 catcagcacc tgccactgccactgtccccc tggctacacg ggcagatact 900 gccaagtgag gtgcagcctg cagtgtgtgcacggccggtt ccgggaggag 950 gagtgctcgt gcgtctgtga catcggctac gggggagcccagtgtgccac 1000 caaggtgcat tttcccttcc acacctgtga cctgaggatc gacggagact1050 gcttcatggt gtcttcagag gcagacacct attacagagc caggatgaaa 1100tgtcagagga aaggcggggt gctggcccag atcaagagcc agaaagtgca 1150 ggacatcctcgccttctatc tgggccgcct ggagaccacc aacgaggtga 1200 ctgacagtga cttcgagaccaggaacttct ggatcgggct cacctacaag 1250 accgccaagg actccttccg ctgggccacaggggagcacc aggccttcac 1300 cagttttgcc tttgggcagc ctgacaacca cgggctggtgtggctgagtg 1350 ctgccatggg gtttggcaac tgcgtggagc tgcaggcttc agctgccttc1400 aactggaacg accagcgctg caaaacccga aaccgttaca tctgccagtt 1450tgcccaggag cacatctccc ggtggggccc agggtcctga ggcctgacca 1500 catggctccctcgcctgccc tgggagcacc ggctctgctt acctgtctgc 1550 ccacctgtct ggaacaagggccaggttaag accacatgcc tcatgtccaa 1600 agaggtctca gaccttgcac aatgccagaagttgggcaga gagaggcagg 1650 gaggccagtg agggccaggg agtgagtgtt agaagaagctggggcccttc 1700 gcctgctttt gattgggaag atgggcttca attagatggc gaaggagagg1750 acaccgccag tggtccaaaa aggctgctct cttccacctg gcccagaccc 1800tgtggggcag cggagcttcc ctgtggcatg aaccccacgg ggtattaaat 1850 tatgaatcagctgaaaaaaa aaaaaa 1876 50 455 PRT Homo Sapien 50 Met Leu His Pro Glu ThrSer Pro Gly Arg Gly His Leu Leu Ala 1 5 10 15 Val Leu Leu Ala Leu LeuGly Thr Thr Trp Ala Glu Val Trp Pro 20 25 30 Pro Gln Leu Gln Glu Gln AlaPro Met Ala Gly Ala Leu Asn Arg 35 40 45 Lys Glu Ser Phe Leu Leu Leu SerLeu His Asn Arg Leu Arg Ser 50 55 60 Trp Val Gln Pro Pro Ala Ala Asp MetArg Arg Leu Asp Trp Ser 65 70 75 Asp Ser Leu Ala Gln Leu Ala Gln Ala ArgAla Ala Leu Cys Gly 80 85 90 Ile Pro Thr Pro Ser Leu Ala Ser Gly Leu TrpArg Thr Leu Gln 95 100 105 Val Gly Trp Asn Met Gln Leu Leu Pro Ala GlyLeu Ala Ser Phe 110 115 120 Val Glu Val Val Ser Leu Trp Phe Ala Glu GlyGln Arg Tyr Ser 125 130 135 His Ala Ala Gly Glu Cys Ala Arg Asn Ala ThrCys Thr His Tyr 140 145 150 Thr Gln Leu Val Trp Ala Thr Ser Ser Gln LeuGly Cys Gly Arg 155 160 165 His Leu Cys Ser Ala Gly Gln Thr Ala Ile GluAla Phe Val Cys 170 175 180 Ala Tyr Ser Pro Gly Gly Asn Trp Glu Val AsnGly Lys Thr Ile 185 190 195 Ile Pro Tyr Lys Lys Gly Ala Trp Cys Ser LeuCys Thr Ala Ser 200 205 210 Val Ser Gly Cys Phe Lys Ala Trp Asp His AlaGly Gly Leu Cys 215 220 225 Glu Val Pro Arg Asn Pro Cys Arg Met Ser CysGln Asn His Gly 230 235 240 Arg Leu Asn Ile Ser Thr Cys His Cys His CysPro Pro Gly Tyr 245 250 255 Thr Gly Arg Tyr Cys Gln Val Arg Cys Ser LeuGln Cys Val His 260 265 270 Gly Arg Phe Arg Glu Glu Glu Cys Ser Cys ValCys Asp Ile Gly 275 280 285 Tyr Gly Gly Ala Gln Cys Ala Thr Lys Val HisPhe Pro Phe His 290 295 300 Thr Cys Asp Leu Arg Ile Asp Gly Asp Cys PheMet Val Ser Ser 305 310 315 Glu Ala Asp Thr Tyr Tyr Arg Ala Arg Met LysCys Gln Arg Lys 320 325 330 Gly Gly Val Leu Ala Gln Ile Lys Ser Gln LysVal Gln Asp Ile 335 340 345 Leu Ala Phe Tyr Leu Gly Arg Leu Glu Thr ThrAsn Glu Val Thr 350 355 360 Asp Ser Asp Phe Glu Thr Arg Asn Phe Trp IleGly Leu Thr Tyr 365 370 375 Lys Thr Ala Lys Asp Ser Phe Arg Trp Ala ThrGly Glu His Gln 380 385 390 Ala Phe Thr Ser Phe Ala Phe Gly Gln Pro AspAsn His Gly Leu 395 400 405 Val Trp Leu Ser Ala Ala Met Gly Phe Gly AsnCys Val Glu Leu 410 415 420 Gln Ala Ser Ala Ala Phe Asn Trp Asn Asp GlnArg Cys Lys Thr 425 430 435 Arg Asn Arg Tyr Ile Cys Gln Phe Ala Gln GluHis Ile Ser Arg 440 445 450 Trp Gly Pro Gly Ser 455 51 24 DNA ArtificialSequence Synthetic oligonucleotide probe 51 aggaacttct ggatcgggct cacc24 52 24 DNA Artificial Sequence Synthetic oligonucleotide probe 52gggtctgggc caggtggaag agag 24 53 45 DNA Artificial Sequence Syntheticoligonucleotide probe 53 gccaaggact ccttccgctg ggccacaggg gagcaccaggccttc 45 54 2331 DNA Homo Sapien 54 cggacgcgtg ggctgggcgc tgcaaagcgtgtcccgccgg gtccccgagc 50 gtcccgcgcc ctcgccccgc catgctcctg ctgctggggctgtgcctggg 100 gctgtccctg tgtgtggggt cgcaggaaga ggcgcagagc tggggccact150 cttcggagca ggatggactc agggtcccga ggcaagtcag actgttgcag 200aggctgaaaa ccaaaccttt gatgacagaa ttctcagtga agtctaccat 250 catttcccgttatgccttca ctacggtttc ctgcagaatg ctgaacagag 300 cttctgaaga ccaggacattgagttccaga tgcagattcc agctgcagct 350 ttcatcacca acttcactat gcttattggagacaaggtgt atcagggcga 400 aattacagag agagaaaaga agagtggtga tagggtaaaagagaaaagga 450 ataaaaccac agaagaaaat ggagagaagg ggactgaaat attcagagct500 tctgcagtga ttcccagcaa ggacaaagcc gcctttttcc tgagttatga 550ggagcttctg cagaggcgcc tgggcaagta cgagcacagc atcagcgtgc 600 ggccccagcagctgtccggg aggctgagcg tggacgtgaa tatcctggag 650 agcgcgggca tcgcatccctggaggtgctg ccgcttcaca acagcaggca 700 gaggggcagt gggcgcgggg aagatgattctgggcctccc ccatctactg 750 tcattaacca aaatgaaaca tttgccaaca taatttttaaacctactgta 800 gtacaacaag ccaggattgc ccagaatgga attttgggag actttatcat850 tagatatgac gtcaatagag aacagagcat tggggacatc caggttctaa 900atggctattt tgtgcactac tttgctccta aagaccttcc tcctttaccc 950 aagaatgtggtattcgtgct tgacagcagt gcttctatgg tgggaaccaa 1000 actccggcag accaaggatgccctcttcac aattctccat gacctccgac 1050 cccaggaccg tttcagtatc attggattttccaaccggat caaagtatgg 1100 aaggaccact tgatatcagt cactccagac agcatcagggatgggaaagt 1150 gtacattcac catatgtcac ccactggagg cacagacatc aacggggccc1200 tgcagagggc catcaggctc ctcaacaagt acgtggccca cagtggcatt 1250ggagaccgga gcgtgtccct catcgtcttc ctgacggatg ggaagcccac 1300 ggtcggggagacgcacaccc tcaagatcct caacaacacc cgagaggccg 1350 cccgaggcca agtctgcatcttcaccattg gcatcggcaa cgacgtggac 1400 ttcaggctgc tggagaaact gtcgctggagaactgtggcc tcacacggcg 1450 cgtgcacgag gaggaggacg caggctcgca gctcatcgggttctacgatg 1500 aaatcaggac cccgctcctc tctgacatcc gcatcgatta tccccccagc1550 tcagtggtgc aggccaccaa gaccctgttc cccaactact tcaacggctc 1600ggagatcatc attgcgggga agctggtgga caggaagctg gatcacctgc 1650 acgtggaggtcaccgccagc aacagtaaga aattcatcat cctgaagaca 1700 gatgtgcctg tgcggcctcagaaggcaggg aaagatgtca caggaagccc 1750 caggcctgga ggcgatggag agggggacaccaaccacatc gagcgtctct 1800 ggagctacct caccacaaag gagctgctga gctcctggctgcaaagtgac 1850 gatgaaccgg agaaggagcg gctgcggcag cgggcccagg ccctggctgt1900 gagctaccgc ttcctcactc ccttcacctc catgaagctg agggggccgg 1950tcccacgcat ggatggcctg gaggaggccc acggcatgtc ggctgccatg 2000 ggacccgaaccggtggtgca gagcgtgcga ggagctggca cgcagccagg 2050 acctttgctc aagaagccaaactccgtcaa aaaaaaacaa aacaaaacaa 2100 aaaaaagaca tgggagagat ggtgtttttcctctccacca cctggggata 2150 cgatgagaag atggccacct gcaagccagg aagacggccctcaccagaca 2200 ccatgtctgc tggcaccttg atcttggacc tcccagcctc cagaactgtg2250 agaaataaat gtgttttgtt taagctaaaa aaaaaaaaaa aaaaaaaaaa 2300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2331 55 694 PRT Homo Sapien 55 MetLeu Leu Leu Leu Gly Leu Cys Leu Gly Leu Ser Leu Cys Val 1 5 10 15 GlySer Gln Glu Glu Ala Gln Ser Trp Gly His Ser Ser Glu Gln 20 25 30 Asp GlyLeu Arg Val Pro Arg Gln Val Arg Leu Leu Gln Arg Leu 35 40 45 Lys Thr LysPro Leu Met Thr Glu Phe Ser Val Lys Ser Thr Ile 50 55 60 Ile Ser Arg TyrAla Phe Thr Thr Val Ser Cys Arg Met Leu Asn 65 70 75 Arg Ala Ser Glu AspGln Asp Ile Glu Phe Gln Met Gln Ile Pro 80 85 90 Ala Ala Ala Phe Ile ThrAsn Phe Thr Met Leu Ile Gly Asp Lys 95 100 105 Val Tyr Gln Gly Glu IleThr Glu Arg Glu Lys Lys Ser Gly Asp 110 115 120 Arg Val Lys Glu Lys ArgAsn Lys Thr Thr Glu Glu Asn Gly Glu 125 130 135 Lys Gly Thr Glu Ile PheArg Ala Ser Ala Val Ile Pro Ser Lys 140 145 150 Asp Lys Ala Ala Phe PheLeu Ser Tyr Glu Glu Leu Leu Gln Arg 155 160 165 Arg Leu Gly Lys Tyr GluHis Ser Ile Ser Val Arg Pro Gln Gln 170 175 180 Leu Ser Gly Arg Leu SerVal Asp Val Asn Ile Leu Glu Ser Ala 185 190 195 Gly Ile Ala Ser Leu GluVal Leu Pro Leu His Asn Ser Arg Gln 200 205 210 Arg Gly Ser Gly Arg GlyGlu Asp Asp Ser Gly Pro Pro Pro Ser 215 220 225 Thr Val Ile Asn Gln AsnGlu Thr Phe Ala Asn Ile Ile Phe Lys 230 235 240 Pro Thr Val Val Gln GlnAla Arg Ile Ala Gln Asn Gly Ile Leu 245 250 255 Gly Asp Phe Ile Ile ArgTyr Asp Val Asn Arg Glu Gln Ser Ile 260 265 270 Gly Asp Ile Gln Val LeuAsn Gly Tyr Phe Val His Tyr Phe Ala 275 280 285 Pro Lys Asp Leu Pro ProLeu Pro Lys Asn Val Val Phe Val Leu 290 295 300 Asp Ser Ser Ala Ser MetVal Gly Thr Lys Leu Arg Gln Thr Lys 305 310 315 Asp Ala Leu Phe Thr IleLeu His Asp Leu Arg Pro Gln Asp Arg 320 325 330 Phe Ser Ile Ile Gly PheSer Asn Arg Ile Lys Val Trp Lys Asp 335 340 345 His Leu Ile Ser Val ThrPro Asp Ser Ile Arg Asp Gly Lys Val 350 355 360 Tyr Ile His His Met SerPro Thr Gly Gly Thr Asp Ile Asn Gly 365 370 375 Ala Leu Gln Arg Ala IleArg Leu Leu Asn Lys Tyr Val Ala His 380 385 390 Ser Gly Ile Gly Asp ArgSer Val Ser Leu Ile Val Phe Leu Thr 395 400 405 Asp Gly Lys Pro Thr ValGly Glu Thr His Thr Leu Lys Ile Leu 410 415 420 Asn Asn Thr Arg Glu AlaAla Arg Gly Gln Val Cys Ile Phe Thr 425 430 435 Ile Gly Ile Gly Asn AspVal Asp Phe Arg Leu Leu Glu Lys Leu 440 445 450 Ser Leu Glu Asn Cys GlyLeu Thr Arg Arg Val His Glu Glu Glu 455 460 465 Asp Ala Gly Ser Gln LeuIle Gly Phe Tyr Asp Glu Ile Arg Thr 470 475 480 Pro Leu Leu Ser Asp IleArg Ile Asp Tyr Pro Pro Ser Ser Val 485 490 495 Val Gln Ala Thr Lys ThrLeu Phe Pro Asn Tyr Phe Asn Gly Ser 500 505 510 Glu Ile Ile Ile Ala GlyLys Leu Val Asp Arg Lys Leu Asp His 515 520 525 Leu His Val Glu Val ThrAla Ser Asn Ser Lys Lys Phe Ile Ile 530 535 540 Leu Lys Thr Asp Val ProVal Arg Pro Gln Lys Ala Gly Lys Asp 545 550 555 Val Thr Gly Ser Pro ArgPro Gly Gly Asp Gly Glu Gly Asp Thr 560 565 570 Asn His Ile Glu Arg LeuTrp Ser Tyr Leu Thr Thr Lys Glu Leu 575 580 585 Leu Ser Ser Trp Leu GlnSer Asp Asp Glu Pro Glu Lys Glu Arg 590 595 600 Leu Arg Gln Arg Ala GlnAla Leu Ala Val Ser Tyr Arg Phe Leu 605 610 615 Thr Pro Phe Thr Ser MetLys Leu Arg Gly Pro Val Pro Arg Met 620 625 630 Asp Gly Leu Glu Glu AlaHis Gly Met Ser Ala Ala Met Gly Pro 635 640 645 Glu Pro Val Val Gln SerVal Arg Gly Ala Gly Thr Gln Pro Gly 650 655 660 Pro Leu Leu Lys Lys ProAsn Ser Val Lys Lys Lys Gln Asn Lys 665 670 675 Thr Lys Lys Arg His GlyArg Asp Gly Val Phe Pro Leu His His 680 685 690 Leu Gly Ile Arg 56 24DNA Artificial Sequence Synthetic oligonucleotide probe 56 gtgggaaccaaactccggca gacc 24 57 18 DNA Artificial Sequence Syntheticoligonucleotide probe 57 cacatcgagc gtctctgg 18 58 24 DNA ArtificialSequence Synthetic oligonucleotide probe 58 agccgctcct tctccggttc atcg24 59 48 DNA Artificial Sequence Synthetic oligonucleotide probe 59tggaaggacc acttgatatc agtcactcca gacagcatca gggatggg 48 60 1413 DNA HomoSapien 60 cggacgcgtg gggtgcccga catggcgagt gtagtgctgc cgagcggatc 50ccagtgtgcg gcggcagcgg cggcggcggc gcctcccggg ctccggcttc 100 tgctgttgctcttctccgcc gcggcactga tccccacagg tgatgggcag 150 aatctgttta cgaaagacgtgacagtgatc gagggagagg ttgcgaccat 200 cagttgccaa gtcaataaga gtgacgactctgtgattcag ctactgaatc 250 ccaacaggca gaccatttat ttcagggact tcaggcctttgaaggacagc 300 aggtttcagt tgctgaattt ttctagcagt gaactcaaag tatcattgac350 aaacgtctca atttctgatg aaggaagata cttttgccag ctctataccg 400atcccccaca ggaaagttac accaccatca cagtcctggt cccaccacgt 450 aatctgatgatcgatatcca gaaagacact gcggtggaag gtgaggagat 500 tgaagtcaac tgcactgctatggccagcaa gccagccacg actatcaggt 550 ggttcaaagg gaacacagag ctaaaaggcaaatcggaggt ggaagagtgg 600 tcagacatgt acactgtgac cagtcagctg atgctgaaggtgcacaagga 650 ggacgatggg gtcccagtga tctgccaggt ggagcaccct gcggtcactg700 gaaacctgca gacccagcgg tatctagaag tacagtataa gcctcaagtg 750cacattcaga tgacttatcc tctacaaggc ttaacccggg aaggggacgc 800 gcttgagttaacatgtgaag ccatcgggaa gccccagcct gtgatggtaa 850 cttgggtgag agtcgatgatgaaatgcctc aacacgccgt actgtctggg 900 cccaacctgt tcatcaataa cctaaacaaaacagataatg gtacataccg 950 ctgtgaagct tcaaacatag tggggaaagc tcactcggattatatgctgt 1000 atgtatacga tccccccaca actatccctc ctcccacaac aaccaccacc1050 accaccacca ccaccaccac caccatcctt accatcatca cagattcccg 1100agcaggtgaa gaaggctcga tcagggcagt ggatcatgcc gtgatcggtg 1150 gcgtcgtggcggtggtggtg ttcgccatgc tgtgcttgct catcattctg 1200 gggcgctatt ttgccagacataaaggtaca tacttcactc atgaagccaa 1250 aggagccgat gacgcagcag acgcagacacagctataatc aatgcagaag 1300 gaggacagaa caactccgaa gaaaagaaag agtacttcatctagatcagc 1350 ctttttgttt caatgaggtg tccaactggc cctatttaga tgataaagag1400 acagtgatat tgg 1413 61 440 PRT Homo Sapien 61 Met Ala Ser Val ValLeu Pro Ser Gly Ser Gln Cys Ala Ala Ala 1 5 10 15 Ala Ala Ala Ala AlaPro Pro Gly Leu Arg Leu Leu Leu Leu Leu 20 25 30 Phe Ser Ala Ala Ala LeuIle Pro Thr Gly Asp Gly Gln Asn Leu 35 40 45 Phe Thr Lys Asp Val Thr ValIle Glu Gly Glu Val Ala Thr Ile 50 55 60 Ser Cys Gln Val Asn Lys Ser AspAsp Ser Val Ile Gln Leu Leu 65 70 75 Asn Pro Asn Arg Gln Thr Ile Tyr PheArg Asp Phe Arg Pro Leu 80 85 90 Lys Asp Ser Arg Phe Gln Leu Leu Asn PheSer Ser Ser Glu Leu 95 100 105 Lys Val Ser Leu Thr Asn Val Ser Ile SerAsp Glu Gly Arg Tyr 110 115 120 Phe Cys Gln Leu Tyr Thr Asp Pro Pro GlnGlu Ser Tyr Thr Thr 125 130 135 Ile Thr Val Leu Val Pro Pro Arg Asn LeuMet Ile Asp Ile Gln 140 145 150 Lys Asp Thr Ala Val Glu Gly Glu Glu IleGlu Val Asn Cys Thr 155 160 165 Ala Met Ala Ser Lys Pro Ala Thr Thr IleArg Trp Phe Lys Gly 170 175 180 Asn Thr Glu Leu Lys Gly Lys Ser Glu ValGlu Glu Trp Ser Asp 185 190 195 Met Tyr Thr Val Thr Ser Gln Leu Met LeuLys Val His Lys Glu 200 205 210 Asp Asp Gly Val Pro Val Ile Cys Gln ValGlu His Pro Ala Val 215 220 225 Thr Gly Asn Leu Gln Thr Gln Arg Tyr LeuGlu Val Gln Tyr Lys 230 235 240 Pro Gln Val His Ile Gln Met Thr Tyr ProLeu Gln Gly Leu Thr 245 250 255 Arg Glu Gly Asp Ala Leu Glu Leu Thr CysGlu Ala Ile Gly Lys 260 265 270 Pro Gln Pro Val Met Val Thr Trp Val ArgVal Asp Asp Glu Met 275 280 285 Pro Gln His Ala Val Leu Ser Gly Pro AsnLeu Phe Ile Asn Asn 290 295 300 Leu Asn Lys Thr Asp Asn Gly Thr Tyr ArgCys Glu Ala Ser Asn 305 310 315 Ile Val Gly Lys Ala His Ser Asp Tyr MetLeu Tyr Val Tyr Asp 320 325 330 Pro Pro Thr Thr Ile Pro Pro Pro Thr ThrThr Thr Thr Thr Thr 335 340 345 Thr Thr Thr Thr Thr Thr Ile Leu Thr IleIle Thr Asp Ser Arg 350 355 360 Ala Gly Glu Glu Gly Ser Ile Arg Ala ValAsp His Ala Val Ile 365 370 375 Gly Gly Val Val Ala Val Val Val Phe AlaMet Leu Cys Leu Leu 380 385 390 Ile Ile Leu Gly Arg Tyr Phe Ala Arg HisLys Gly Thr Tyr Phe 395 400 405 Thr His Glu Ala Lys Gly Ala Asp Asp AlaAla Asp Ala Asp Thr 410 415 420 Ala Ile Ile Asn Ala Glu Gly Gly Gln AsnAsn Ser Glu Glu Lys 425 430 435 Lys Glu Tyr Phe Ile 440 62 24 DNAArtificial Sequence Synthetic oligonucleotide probe 62 ggcttctgctgttgctcttc tccg 24 63 20 DNA Artificial Sequence Syntheticoligonucleotide probe 63 gtacactgtg accagtcagc 20 64 20 DNA ArtificialSequence Synthetic oligonucleotide probe 64 atcatcacag attcccgagc 20 6524 DNA Artificial Sequence Synthetic oligonucleotide probe 65 ttcaatctcctcaccttcca ccgc 24 66 24 DNA Artificial Sequence Syntheticoligonucleotide probe 66 atagctgtgt ctgcgtctgc tgcg 24 67 50 DNAArtificial Sequence Synthetic oligonucleotide probe 67 cgcggcactgatccccacag gtgatgggca gaatctgttt acgaaagacg 50 68 2555 DNA Homo Sapien68 ggggcgggtg gacgcggact cgaacgcagt tgcttcggga cccaggaccc 50 cctcgggcccgacccgccag gaaagactga ggccgcggcc tgccccgccc 100 ggctccctgc gccgccgccgcctcccggga cagaagatgt gctccagggt 150 ccctctgctg ctgccgctgc tcctgctactggccctgggg cctggggtgc 200 agggctgccc atccggctgc cagtgcagcc agccacagacagtcttctgc 250 actgcccgcc aggggaccac ggtgccccga gacgtgccac ccgacacggt300 ggggctgtac gtctttgaga acggcatcac catgctcgac gcaagcagct 350ttgccggcct gccgggcctg cagctcctgg acctgtcaca gaaccagatc 400 gccagcctgcgcctgccccg cctgctgctg ctggacctca gccacaacag 450 cctcctggcc ctggagcccggcatcctgga cactgccaac gtggaggcgc 500 tgcggctggc tggtctgggg ctgcagcagctggacgaggg gctcttcagc 550 cgcttgcgca acctccacga cctggatgtg tccgacaaccagctggagcg 600 agtgccacct gtgatccgag gcctccgggg cctgacgcgc ctgcggctgg650 ccggcaacac ccgcattgcc cagctgcggc ccgaggacct ggccggcctg 700gctgccctgc aggagctgga tgtgagcaac ctaagcctgc aggccctgcc 750 tggcgacctctcgggcctct tcccccgcct gcggctgctg gcagctgccc 800 gcaacccctt caactgcgtgtgccccctga gctggtttgg cccctgggtg 850 cgcgagagcc acgtcacact ggccagccctgaggagacgc gctgccactt 900 cccgcccaag aacgctggcc ggctgctcct ggagcttgactacgccgact 950 ttggctgccc agccaccacc accacagcca cagtgcccac cacgaggccc1000 gtggtgcggg agcccacagc cttgtcttct agcttggctc ctacctggct 1050tagccccaca gcgccggcca ctgaggcccc cagcccgccc tccactgccc 1100 caccgactgtagggcctgtc ccccagcccc aggactgccc accgtccacc 1150 tgcctcaatg ggggcacatgccacctgggg acacggcacc acctggcgtg 1200 cttgtgcccc gaaggcttca cgggcctgtactgtgagagc cagatggggc 1250 aggggacacg gcccagccct acaccagtca cgccgaggccaccacggtcc 1300 ctgaccctgg gcatcgagcc ggtgagcccc acctccctgc gcgtggggct1350 gcagcgctac ctccagggga gctccgtgca gctcaggagc ctccgtctca 1400cctatcgcaa cctatcgggc cctgataagc ggctggtgac gctgcgactg 1450 cctgcctcgctcgctgagta cacggtcacc cagctgcggc ccaacgccac 1500 ttactccgtc tgtgtcatgcctttggggcc cgggcgggtg ccggagggcg 1550 aggaggcctg cggggaggcc catacacccccagccgtcca ctccaaccac 1600 gccccagtca cccaggcccg cgagggcaac ctgccgctcctcattgcgcc 1650 cgccctggcc gcggtgctcc tggccgcgct ggctgcggtg ggggcagcct1700 actgtgtgcg gcgggggcgg gccatggcag cagcggctca ggacaaaggg 1750caggtggggc caggggctgg gcccctggaa ctggagggag tgaaggtccc 1800 cttggagccaggcccgaagg caacagaggg cggtggagag gccctgccca 1850 gcgggtctga gtgtgaggtgccactcatgg gcttcccagg gcctggcctc 1900 cagtcacccc tccacgcaaa gccctacatctaagccagag agagacaggg 1950 cagctggggc cgggctctca gccagtgaga tggccagccccctcctgctg 2000 ccacaccacg taagttctca gtcccaacct cggggatgtg tgcagacagg2050 gctgtgtgac cacagctggg ccctgttccc tctggacctc ggtctcctca 2100tctgtgagat gctgtggccc agctgacgag ccctaacgtc cccagaaccg 2150 agtgcctatgaggacagtgt ccgccctgcc ctccgcaacg tgcagtccct 2200 gggcacggcg ggccctgccatgtgctggta acgcatgcct gggccctgct 2250 gggctctccc actccaggcg gaccctgggggccagtgaag gaagctcccg 2300 gaaagagcag agggagagcg ggtaggcggc tgtgtgactctagtcttggc 2350 cccaggaagc gaaggaacaa aagaaactgg aaaggaagat gctttaggaa2400 catgttttgc ttttttaaaa tatatatata tttataagag atcctttccc 2450atttattctg ggaagatgtt tttcaaactc agagacaagg actttggttt 2500 ttgtaagacaaacgatgata tgaaggcctt ttgtaagaaa aaataaaaaa 2550 aaaaa 2555 69 598 PRTHomo Sapien 69 Met Cys Ser Arg Val Pro Leu Leu Leu Pro Leu Leu Leu LeuLeu 1 5 10 15 Ala Leu Gly Pro Gly Val Gln Gly Cys Pro Ser Gly Cys GlnCys 20 25 30 Ser Gln Pro Gln Thr Val Phe Cys Thr Ala Arg Gln Gly Thr Thr35 40 45 Val Pro Arg Asp Val Pro Pro Asp Thr Val Gly Leu Tyr Val Phe 5055 60 Glu Asn Gly Ile Thr Met Leu Asp Ala Ser Ser Phe Ala Gly Leu 65 7075 Pro Gly Leu Gln Leu Leu Asp Leu Ser Gln Asn Gln Ile Ala Ser 80 85 90Leu Arg Leu Pro Arg Leu Leu Leu Leu Asp Leu Ser His Asn Ser 95 100 105Leu Leu Ala Leu Glu Pro Gly Ile Leu Asp Thr Ala Asn Val Glu 110 115 120Ala Leu Arg Leu Ala Gly Leu Gly Leu Gln Gln Leu Asp Glu Gly 125 130 135Leu Phe Ser Arg Leu Arg Asn Leu His Asp Leu Asp Val Ser Asp 140 145 150Asn Gln Leu Glu Arg Val Pro Pro Val Ile Arg Gly Leu Arg Gly 155 160 165Leu Thr Arg Leu Arg Leu Ala Gly Asn Thr Arg Ile Ala Gln Leu 170 175 180Arg Pro Glu Asp Leu Ala Gly Leu Ala Ala Leu Gln Glu Leu Asp 185 190 195Val Ser Asn Leu Ser Leu Gln Ala Leu Pro Gly Asp Leu Ser Gly 200 205 210Leu Phe Pro Arg Leu Arg Leu Leu Ala Ala Ala Arg Asn Pro Phe 215 220 225Asn Cys Val Cys Pro Leu Ser Trp Phe Gly Pro Trp Val Arg Glu 230 235 240Ser His Val Thr Leu Ala Ser Pro Glu Glu Thr Arg Cys His Phe 245 250 255Pro Pro Lys Asn Ala Gly Arg Leu Leu Leu Glu Leu Asp Tyr Ala 260 265 270Asp Phe Gly Cys Pro Ala Thr Thr Thr Thr Ala Thr Val Pro Thr 275 280 285Thr Arg Pro Val Val Arg Glu Pro Thr Ala Leu Ser Ser Ser Leu 290 295 300Ala Pro Thr Trp Leu Ser Pro Thr Ala Pro Ala Thr Glu Ala Pro 305 310 315Ser Pro Pro Ser Thr Ala Pro Pro Thr Val Gly Pro Val Pro Gln 320 325 330Pro Gln Asp Cys Pro Pro Ser Thr Cys Leu Asn Gly Gly Thr Cys 335 340 345His Leu Gly Thr Arg His His Leu Ala Cys Leu Cys Pro Glu Gly 350 355 360Phe Thr Gly Leu Tyr Cys Glu Ser Gln Met Gly Gln Gly Thr Arg 365 370 375Pro Ser Pro Thr Pro Val Thr Pro Arg Pro Pro Arg Ser Leu Thr 380 385 390Leu Gly Ile Glu Pro Val Ser Pro Thr Ser Leu Arg Val Gly Leu 395 400 405Gln Arg Tyr Leu Gln Gly Ser Ser Val Gln Leu Arg Ser Leu Arg 410 415 420Leu Thr Tyr Arg Asn Leu Ser Gly Pro Asp Lys Arg Leu Val Thr 425 430 435Leu Arg Leu Pro Ala Ser Leu Ala Glu Tyr Thr Val Thr Gln Leu 440 445 450Arg Pro Asn Ala Thr Tyr Ser Val Cys Val Met Pro Leu Gly Pro 455 460 465Gly Arg Val Pro Glu Gly Glu Glu Ala Cys Gly Glu Ala His Thr 470 475 480Pro Pro Ala Val His Ser Asn His Ala Pro Val Thr Gln Ala Arg 485 490 495Glu Gly Asn Leu Pro Leu Leu Ile Ala Pro Ala Leu Ala Ala Val 500 505 510Leu Leu Ala Ala Leu Ala Ala Val Gly Ala Ala Tyr Cys Val Arg 515 520 525Arg Gly Arg Ala Met Ala Ala Ala Ala Gln Asp Lys Gly Gln Val 530 535 540Gly Pro Gly Ala Gly Pro Leu Glu Leu Glu Gly Val Lys Val Pro 545 550 555Leu Glu Pro Gly Pro Lys Ala Thr Glu Gly Gly Gly Glu Ala Leu 560 565 570Pro Ser Gly Ser Glu Cys Glu Val Pro Leu Met Gly Phe Pro Gly 575 580 585Pro Gly Leu Gln Ser Pro Leu His Ala Lys Pro Tyr Ile 590 595 70 22 DNAArtificial Sequence Synthetic oligonucleotide probe 70 ccctccactgccccaccgac tg 22 71 24 DNA Artificial Sequence Synthetic oligonucleotideprobe 71 cggttctggg gacgttaggg ctcg 24 72 25 DNA Artificial SequenceSynthetic oligonucleotide probe 72 ctgcccaccg tccacctgcc tcaat 25 73 45DNA Artificial Sequence Synthetic oligonucleotide probe 73 aggactgcccaccgtccacc tgcctcaatg ggggcacatg ccacc 45 74 45 DNA Artificial SequenceSynthetic Oligonucleotide Probe 74 acgcaaagcc ctacatctaa gccagagagagacagggcag ctggg 45 75 1077 DNA Homo Sapien 75 ggcactagga caaccttcttcccttctgca ccactgcccg tacccttacc 50 cgccccgcca cctccttgct accccactcttgaaaccaca gctgttggca 100 gggtccccag ctcatgccag cctcatctcc tttcttgctagcccccaaag 150 ggcctccagg caacatgggg ggcccagtca gagagccggc actctcagtt200 gccctctggt tgagttgggg ggcagctctg ggggccgtgg cttgtgccat 250ggctctgctg acccaacaaa cagagctgca gagcctcagg agagaggtga 300 gccggctgcaggggacagga ggcccctccc agaatgggga agggtatccc 350 tggcagagtc tcccggagcagagttccgat gccctggaag cctgggagaa 400 tggggagaga tcccggaaaa ggagagcagtgctcacccaa aaacagaaga 450 agcagcactc tgtcctgcac ctggttccca ttaacgccacctccaaggat 500 gactccgatg tgacagaggt gatgtggcaa ccagctctta ggcgtgggag550 aggcctacag gcccaaggat atggtgtccg aatccaggat gctggagttt 600atctgctgta tagccaggtc ctgtttcaag acgtgacttt caccatgggt 650 caggtggtgtctcgagaagg ccaaggaagg caggagactc tattccgatg 700 tataagaagt atgccctcccacccggaccg ggcctacaac agctgctata 750 gcgcaggtgt cttccattta caccaaggggatattctgag tgtcataatt 800 ccccgggcaa gggcgaaact taacctctct ccacatggaaccttcctggg 850 gtttgtgaaa ctgtgattgt gttataaaaa gtggctccca gcttggaaga900 ccagggtggg tacatactgg agacagccaa gagctgagta tataaaggag 950agggaatgtg caggaacaga ggcatcttcc tgggtttggc tccccgttcc 1000 tcacttttcccttttcattc ccacccccta gactttgatt ttacggatat 1050 cttgcttctg ttccccatggagctccg 1077 76 250 PRT Homo Sapien 76 Met Pro Ala Ser Ser Pro Phe LeuLeu Ala Pro Lys Gly Pro Pro 1 5 10 15 Gly Asn Met Gly Gly Pro Val ArgGlu Pro Ala Leu Ser Val Ala 20 25 30 Leu Trp Leu Ser Trp Gly Ala Ala LeuGly Ala Val Ala Cys Ala 35 40 45 Met Ala Leu Leu Thr Gln Gln Thr Glu LeuGln Ser Leu Arg Arg 50 55 60 Glu Val Ser Arg Leu Gln Gly Thr Gly Gly ProSer Gln Asn Gly 65 70 75 Glu Gly Tyr Pro Trp Gln Ser Leu Pro Glu Gln SerSer Asp Ala 80 85 90 Leu Glu Ala Trp Glu Asn Gly Glu Arg Ser Arg Lys ArgArg Ala 95 100 105 Val Leu Thr Gln Lys Gln Lys Lys Gln His Ser Val LeuHis Leu 110 115 120 Val Pro Ile Asn Ala Thr Ser Lys Asp Asp Ser Asp ValThr Glu 125 130 135 Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg Gly LeuGln Ala 140 145 150 Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val TyrLeu Leu 155 160 165 Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr MetGly Gln 170 175 180 Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr LeuPhe Arg 185 190 195 Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala TyrAsn Ser 200 205 210 Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly AspIle Leu 215 220 225 Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn LeuSer Pro 230 235 240 His Gly Thr Phe Leu Gly Phe Val Lys Leu 245 250 772849 DNA Homo Sapien 77 cactttctcc ctctcttcct ttactttcga gaaaccgcgcttccgcttct 50 ggtcgcagag acctcggaga ccgcgccggg gagacggagg tgctgtgggt 100gggggggacc tgtggctgct cgtaccgccc cccaccctcc tcttctgcac 150 tgccgtcctccggaagacct tttcccctgc tctgtttcct tcaccgagtc 200 tgtgcatcgc cccggacctggccgggagga ggcttggccg gcgggagatg 250 ctctaggggc ggcgcgggag gagcggccggcgggacggag ggcccggcag 300 gaagatgggc tcccgtggac agggactctt gctggcgtactgcctgctcc 350 ttgcctttgc ctctggcctg gtcctgagtc gtgtgcccca tgtccagggg400 gaacagcagg agtgggaggg gactgaggag ctgccgtcgc ctccggacca 450tgccgagagg gctgaagaac aacatgaaaa atacaggccc agtcaggacc 500 aggggctccctgcttcccgg tgcttgcgct gctgtgaccc cggtacctcc 550 atgtacccgg cgaccgccgtgccccagatc aacatcacta tcttgaaagg 600 ggagaagggt gaccgcggag atcgaggcctccaagggaaa tatggcaaaa 650 caggctcagc aggggccagg ggccacactg gacccaaagggcagaagggc 700 tccatggggg cccctgggga gcggtgcaag agccactacg ccgccttttc750 ggtgggccgg aagaagccca tgcacagcaa ccactactac cagacggtga 800tcttcgacac ggagttcgtg aacctctacg accacttcaa catgttcacc 850 ggcaagttctactgctacgt gcccggcctc tacttcttca gcctcaacgt 900 gcacacctgg aaccagaaggagacctacct gcacatcatg aagaacgagg 950 aggaggtggt gatcttgttc gcgcaggtgggcgaccgcag catcatgcaa 1000 agccagagcc tgatgctgga gctgcgagag caggaccaggtgtgggtacg 1050 cctctacaag ggcgaacgtg agaacgccat cttcagcgag gagctggaca1100 cctacatcac cttcagtggc tacctggtca agcacgccac cgagccctag 1150ctggccggcc acctcctttc ctctcgccac cttccacccc tgcgctgtgc 1200 tgaccccaccgcctcttccc cgatccctgg actccgactc cctggctttg 1250 gcattcagtg agacgccctgcacacacaga aagccaaagc gatcggtgct 1300 cccagatccc gcagcctctg gagagagctgacggcagatg aaatcaccag 1350 ggcggggcac ccgcgagaac cctctgggac cttccgcggccctctctgca 1400 cacatcctca agtgaccccg cacggcgaga cgcgggtggc ggcagggcgt1450 cccagggtgc ggcaccgcgg ctccagtcct tggaaataat taggcaaatt 1500ctaaaggtct caaaaggagc aaagtaaacc gtggaggaca aagaaaaggg 1550 ttgttatttttgtctttcca gccagcctgc tggctcccaa gagagaggcc 1600 ttttcagttg agactctgcttaagagaaga tccaaagtta aagctctggg 1650 gtcaggggag gggccggggg caggaaactacctctggctt aattctttta 1700 agccacgtag gaactttctt gagggatagg tggaccctgacatccctgtg 1750 gccttgccca agggctctgc tggtctttct gagtcacagc tgcgaggtga1800 tgggggctgg ggccccaggc gtcagcctcc cagagggaca gctgagcccc 1850ctgccttggc tccaggttgg tagaagcagc cgaagggctc ctgacagtgg 1900 ccagggacccctgggtcccc caggcctgca gatgtttcta tgaggggcag 1950 agctccttgg tacatccatgtgtggctctg ctccacccct gtgccacccc 2000 agagccctgg ggggtggtct ccatgcctgccaccctggca tcggctttct 2050 gtgccgcctc ccacacaaat cagccccaga aggccccggggccttggctt 2100 ctgtttttta taaaacacct caagcagcac tgcagtctcc catctcctcg2150 tgggctaagc atcaccgctt ccacgtgtgt tgtgttggtt ggcagcaagg 2200ctgatccaga ccccttctgc ccccactgcc ctcatccagg cctctgacca 2250 gtagcctgagaggggctttt tctaggcttc agagcagggg agagctggaa 2300 ggggctagaa agctcccgcttgtctgtttc tcaggctcct gtgagcctca 2350 gtcctgagac cagagtcaag aggaagtacacgtcccaatc acccgtgtca 2400 ggattcactc tcaggagctg ggtggcagga gaggcaatagcccctgtggc 2450 aattgcagga ccagctggag cagggttgcg gtgtctccac ggtgctctcg2500 ccctgcccat ggccacccca gactctgatc tccaggaacc ccatagcccc 2550tctccacctc accccatgtt gatgcccagg gtcactcttg ctacccgctg 2600 ggcccccaaacccccgctgc ctctcttcct tccccccatc ccccacctgg 2650 ttttgactaa tcctgcttccctctctgggc ctggctgccg ggatctgggg 2700 tccctaagtc cctctcttta aagaacttctgcgggtcaga ctctgaagcc 2750 gagttgctgt gggcgtgccc ggaagcagag cgccacactcgctgcttaag 2800 ctcccccagc tctttccaga aaacattaaa ctcagaattg tgttttcaa2849 78 281 PRT Homo Sapien 78 Met Gly Ser Arg Gly Gln Gly Leu Leu LeuAla Tyr Cys Leu Leu 1 5 10 15 Leu Ala Phe Ala Ser Gly Leu Val Leu SerArg Val Pro His Val 20 25 30 Gln Gly Glu Gln Gln Glu Trp Glu Gly Thr GluGlu Leu Pro Ser 35 40 45 Pro Pro Asp His Ala Glu Arg Ala Glu Glu Gln HisGlu Lys Tyr 50 55 60 Arg Pro Ser Gln Asp Gln Gly Leu Pro Ala Ser Arg CysLeu Arg 65 70 75 Cys Cys Asp Pro Gly Thr Ser Met Tyr Pro Ala Thr Ala ValPro 80 85 90 Gln Ile Asn Ile Thr Ile Leu Lys Gly Glu Lys Gly Asp Arg Gly95 100 105 Asp Arg Gly Leu Gln Gly Lys Tyr Gly Lys Thr Gly Ser Ala Gly110 115 120 Ala Arg Gly His Thr Gly Pro Lys Gly Gln Lys Gly Ser Met Gly125 130 135 Ala Pro Gly Glu Arg Cys Lys Ser His Tyr Ala Ala Phe Ser Val140 145 150 Gly Arg Lys Lys Pro Met His Ser Asn His Tyr Tyr Gln Thr Val155 160 165 Ile Phe Asp Thr Glu Phe Val Asn Leu Tyr Asp His Phe Asn Met170 175 180 Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gly Leu Tyr Phe Phe185 190 195 Ser Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr Leu His200 205 210 Ile Met Lys Asn Glu Glu Glu Val Val Ile Leu Phe Ala Gln Val215 220 225 Gly Asp Arg Ser Ile Met Gln Ser Gln Ser Leu Met Leu Glu Leu230 235 240 Arg Glu Gln Asp Gln Val Trp Val Arg Leu Tyr Lys Gly Glu Arg245 250 255 Glu Asn Ala Ile Phe Ser Glu Glu Leu Asp Thr Tyr Ile Thr Phe260 265 270 Ser Gly Tyr Leu Val Lys His Ala Thr Glu Pro 275 280 79 24DNA Artificial Sequence Synthetic oligonucleotide probe 79 tacaggcccagtcaggacca gggg 24 80 24 DNA Artificial Sequence Syntheticoligonucleotide probe 80 ctgaagaagt agaggccggg cacg 24 81 45 DNAArtificial Sequence Synthetic oligonucleotide probe 81 cccggtgcttgcgctgctgt gaccccggta cctccatgta cccgg 45 82 2284 DNA Homo Sapien 82gcggagcatc cgctgcggtc ctcgccgaga cccccgcgcg gattcgccgg 50 tccttcccgcgggcgcgaca gagctgtcct cgcacctgga tggcagcagg 100 ggcgccgggg tcctctcgacgccagagaga aatctcatca tctgtgcagc 150 cttcttaaag caaactaaga ccagagggaggattatcctt gacctttgaa 200 gaccaaaact aaactgaaat ttaaaatgtt cttcgggggagaagggagct 250 tgacttacac tttggtaata atttgcttcc tgacactaag gctgtctgct300 agtcagaatt gcctcaaaaa gagtctagaa gatgttgtca ttgacatcca 350gtcatctctt tctaagggaa tcagaggcaa tgagcccgta tatacttcaa 400 ctcaagaagactgcattaat tcttgctgtt caacaaaaaa catatcaggg 450 gacaaagcat gtaacttgatgatcttcgac actcgaaaaa cagctagaca 500 acccaactgc tacctatttt tctgtcccaacgaggaagcc tgtccattga 550 aaccagcaaa aggacttatg agttacagga taattacagattttccatct 600 ttgaccagaa atttgccaag ccaagagtta ccccaggaag attctctctt650 acatggccaa ttttcacaag cagtcactcc cctagcccat catcacacag 700attattcaaa gcccaccgat atctcatgga gagacacact ttctcagaag 750 tttggatcctcagatcacct ggagaaacta tttaagatgg atgaagcaag 800 tgcccagctc cttgcttataaggaaaaagg ccattctcag agttcacaat 850 tttcctctga tcaagaaata gctcatctgctgcctgaaaa tgtgagtgcg 900 ctcccagcta cggtggcagt tgcttctcca cataccacctcggctactcc 950 aaagcccgcc acccttctac ccaccaatgc ttcagtgaca ccttctggga1000 cttcccagcc acagctggcc accacagctc cacctgtaac cactgtcact 1050tctcagcctc ccacgaccct catttctaca gtttttacac gggctgcggc 1100 tacactccaagcaatggcta caacagcagt tctgactacc acctttcagg 1150 cacctacgga ctcgaaaggcagcttagaaa ccataccgtt tacagaaatc 1200 tccaacttaa ctttgaacac agggaatgtgtataacccta ctgcactttc 1250 tatgtcaaat gtggagtctt ccactatgaa taaaactgcttcctgggaag 1300 gtagggaggc cagtccaggc agttcctccc agggcagtgt tccagaaaat1350 cagtacggcc ttccatttga aaaatggctt cttatcgggt ccctgctctt 1400tggtgtcctg ttcctggtga taggcctcgt cctcctgggt agaatccttt 1450 cggaatcactccgcaggaaa cgttactcaa gactggatta tttgatcaat 1500 gggatctatg tggacatctaaggatggaac tcggtgtctc ttaattcatt 1550 tagtaaccag aagcccaaat gcaatgagtttctgctgact tgctagtctt 1600 agcaggaggt tgtattttga agacaggaaa atgcccccttctgctttcct 1650 tttttttttt ggagacagag tcttgctctg ttgcccaggc tggagtgcag1700 tagcacgatc tcggctctca ccgcaacctc cgtctcctgg gttcaagcga 1750ttctcctgcc tcagcctcct aagtatctgg gattacaggc atgtgccacc 1800 acacctgggtgatttttgta tttttagtag agacggggtt tcaccatgtt 1850 ggtcaggctg gtctcaaactcctgacctag tgatccaccc tcctcggcct 1900 cccaaagtgc tgggattaca ggcatgagccaccacagctg gcccccttct 1950 gttttatgtt tggtttttga gaaggaatga agtgggaaccaaattaggta 2000 attttgggta atctgtctct aaaatattag ctaaaaacaa agctctatgt2050 aaagtaataa agtataattg ccatataaat ttcaaaattc aactggcttt 2100tatgcaaaga aacaggttag gacatctagg ttccaattca ttcacattct 2150 tggttccagataaaatcaac tgtttatatc aatttctaat ggatttgctt 2200 ttctttttat atggattcctttaaaactta ttccagatgt agttccttcc 2250 aattaaatat ttgaataaat cttttgttactcaa 2284 83 431 PRT Homo Sapien 83 Met Phe Phe Gly Gly Glu Gly Ser LeuThr Tyr Thr Leu Val Ile 1 5 10 15 Ile Cys Phe Leu Thr Leu Arg Leu SerAla Ser Gln Asn Cys Leu 20 25 30 Lys Lys Ser Leu Glu Asp Val Val Ile AspIle Gln Ser Ser Leu 35 40 45 Ser Lys Gly Ile Arg Gly Asn Glu Pro Val TyrThr Ser Thr Gln 50 55 60 Glu Asp Cys Ile Asn Ser Cys Cys Ser Thr Lys AsnIle Ser Gly 65 70 75 Asp Lys Ala Cys Asn Leu Met Ile Phe Asp Thr Arg LysThr Ala 80 85 90 Arg Gln Pro Asn Cys Tyr Leu Phe Phe Cys Pro Asn Glu GluAla 95 100 105 Cys Pro Leu Lys Pro Ala Lys Gly Leu Met Ser Tyr Arg IleIle 110 115 120 Thr Asp Phe Pro Ser Leu Thr Arg Asn Leu Pro Ser Gln GluLeu 125 130 135 Pro Gln Glu Asp Ser Leu Leu His Gly Gln Phe Ser Gln AlaVal 140 145 150 Thr Pro Leu Ala His His His Thr Asp Tyr Ser Lys Pro ThrAsp 155 160 165 Ile Ser Trp Arg Asp Thr Leu Ser Gln Lys Phe Gly Ser SerAsp 170 175 180 His Leu Glu Lys Leu Phe Lys Met Asp Glu Ala Ser Ala GlnLeu 185 190 195 Leu Ala Tyr Lys Glu Lys Gly His Ser Gln Ser Ser Gln PheSer 200 205 210 Ser Asp Gln Glu Ile Ala His Leu Leu Pro Glu Asn Val SerAla 215 220 225 Leu Pro Ala Thr Val Ala Val Ala Ser Pro His Thr Thr SerAla 230 235 240 Thr Pro Lys Pro Ala Thr Leu Leu Pro Thr Asn Ala Ser ValThr 245 250 255 Pro Ser Gly Thr Ser Gln Pro Gln Leu Ala Thr Thr Ala ProPro 260 265 270 Val Thr Thr Val Thr Ser Gln Pro Pro Thr Thr Leu Ile SerThr 275 280 285 Val Phe Thr Arg Ala Ala Ala Thr Leu Gln Ala Met Ala ThrThr 290 295 300 Ala Val Leu Thr Thr Thr Phe Gln Ala Pro Thr Asp Ser LysGly 305 310 315 Ser Leu Glu Thr Ile Pro Phe Thr Glu Ile Ser Asn Leu ThrLeu 320 325 330 Asn Thr Gly Asn Val Tyr Asn Pro Thr Ala Leu Ser Met SerAsn 335 340 345 Val Glu Ser Ser Thr Met Asn Lys Thr Ala Ser Trp Glu GlyArg 350 355 360 Glu Ala Ser Pro Gly Ser Ser Ser Gln Gly Ser Val Pro GluAsn 365 370 375 Gln Tyr Gly Leu Pro Phe Glu Lys Trp Leu Leu Ile Gly SerLeu 380 385 390 Leu Phe Gly Val Leu Phe Leu Val Ile Gly Leu Val Leu LeuGly 395 400 405 Arg Ile Leu Ser Glu Ser Leu Arg Arg Lys Arg Tyr Ser ArgLeu 410 415 420 Asp Tyr Leu Ile Asn Gly Ile Tyr Val Asp Ile 425 430 8430 DNA Artificial Sequence Synthetic oligonucleotide probe 84 agggaggattatccttgacc tttgaagacc 30 85 18 DNA Artificial Sequence Syntheticoligonucleotide probe 85 gaagcaagtg cccagctc 18 86 18 DNA ArtificialSequence Synthetic oligonucleotide probe 86 cgggtccctg ctctttgg 18 87 24DNA Artificial Sequence Synthetic oligonucleotide probe 87 caccgtagctgggagcgcac tcac 24 88 18 DNA Artificial Sequence Syntheticoligonucleotide probe 88 agtgtaagtc aagctccc 18 89 49 DNA ArtificialSequence Synthetic oligonucleotide probe 89 gcttcctgac actaaggctgtctgctagtc agaattgcct caaaaagag 49 90 957 DNA Homo Sapien 90 cctggaagatgcgcccattg gctggtggcc tgctcaaggt ggtgttcgtg 50 gtcttcgcct ccttgtgtgcctggtattcg gggtacctgc tcgcagagct 100 cattccagat gcacccctgt ccagtgctgcctatagcatc cgcagcatcg 150 gggagaggcc tgtcctcaaa gctccagtcc ccaaaaggcaaaaatgtgac 200 cactggactc cctgcccatc tgacacctat gcctacaggt tactcagcgg250 aggtggcaga agcaagtacg ccaaaatctg ctttgaggat aacctactta 300tgggagaaca gctgggaaat gttgccagag gaataaacat tgccattgtc 350 aactatgtaactgggaatgt gacagcaaca cgatgttttg atatgtatga 400 aggcgataac tctggaccgatgacaaagtt tattcagagt gctgctccaa 450 aatccctgct cttcatggtg acctatgacgacggaagcac aagactgaat 500 aacgatgcca agaatgccat agaagcactt ggaagtaaagaaatcaggaa 550 catgaaattc aggtctagct gggtatttat tgcagcaaaa ggcttggaac600 tcccttccga aattcagaga gaaaagatca accactctga tgctaagaac 650aacagatatt ctggctggcc tgcagagatc cagatagaag gctgcatacc 700 caaagaacgaagctgacact gcagggtcct gagtaaatgt gttctgtata 750 aacaaatgca gctggaatcgctcaagaatc ttatttttct aaatccaaca 800 gcccatattt gatgagtatt ttgggtttgttgtaaaccaa tgaacatttg 850 ctagttgtat caaatcttgg tacgcagtat ttttataccagtattttatg 900 tagtgaagat gtcaattagc aggaaactaa aatgaatgga aattcttaaa950 aaaaaaa 957 91 235 PRT Homo Sapien 91 Met Arg Pro Leu Ala Gly GlyLeu Leu Lys Val Val Phe Val Val 1 5 10 15 Phe Ala Ser Leu Cys Ala TrpTyr Ser Gly Tyr Leu Leu Ala Glu 20 25 30 Leu Ile Pro Asp Ala Pro Leu SerSer Ala Ala Tyr Ser Ile Arg 35 40 45 Ser Ile Gly Glu Arg Pro Val Leu LysAla Pro Val Pro Lys Arg 50 55 60 Gln Lys Cys Asp His Trp Thr Pro Cys ProSer Asp Thr Tyr Ala 65 70 75 Tyr Arg Leu Leu Ser Gly Gly Gly Arg Ser LysTyr Ala Lys Ile 80 85 90 Cys Phe Glu Asp Asn Leu Leu Met Gly Glu Gln LeuGly Asn Val 95 100 105 Ala Arg Gly Ile Asn Ile Ala Ile Val Asn Tyr ValThr Gly Asn 110 115 120 Val Thr Ala Thr Arg Cys Phe Asp Met Tyr Glu GlyAsp Asn Ser 125 130 135 Gly Pro Met Thr Lys Phe Ile Gln Ser Ala Ala ProLys Ser Leu 140 145 150 Leu Phe Met Val Thr Tyr Asp Asp Gly Ser Thr ArgLeu Asn Asn 155 160 165 Asp Ala Lys Asn Ala Ile Glu Ala Leu Gly Ser LysGlu Ile Arg 170 175 180 Asn Met Lys Phe Arg Ser Ser Trp Val Phe Ile AlaAla Lys Gly 185 190 195 Leu Glu Leu Pro Ser Glu Ile Gln Arg Glu Lys IleAsn His Ser 200 205 210 Asp Ala Lys Asn Asn Arg Tyr Ser Gly Trp Pro AlaGlu Ile Gln 215 220 225 Ile Glu Gly Cys Ile Pro Lys Glu Arg Ser 230 23592 20 DNA Artificial Sequence Synthetic oligonucleotide probe 92aatgtgacca ctggactccc 20 93 18 DNA Artificial Sequence Syntheticoligonucleotide probe 93 aggcttggaa ctcccttc 18 94 24 DNA ArtificialSequence Synthetic oligonucleotide probe 94 aagattcttg agcgattcca gctg24 95 47 DNA Artificial Sequence Synthetic oligonucleotide probe 95aatccctgct cttcatggtg acctatgacg acggaagcac aagactg 47 96 21 DNAArtificial Sequence Synthetic oligonucleotide probe 96 ctcaagaagcacgcgtactg c 21 97 25 DNA Artificial Sequence Synthetic oligonucleotideprobe 97 ccaacctcag cttccgcctc tacga 25 98 18 DNA Artificial SequenceSynthetic oligonucleotide probe 98 catccaggct cgccactg 18 99 20 DNAArtificial Sequence Synthetic oligonucleotide probe 99 tggcaaggaatgggaacagt 20 100 25 DNA Artificial Sequence Synthetic oligonucleotideprobe 100 atgctgccag acctgatcgc agaca 25 101 19 DNA Artificial SequenceSynthetic oligonucleotide probe 101 gggcagaaat ccagccact 19 102 18 DNAArtificial Sequence Synthetic oligonucleotide probe 102 cccttcgcctgcttttga 18 103 27 DNA Artificial Sequence Synthetic oligonucleotideprobe 103 gccatctaat tgaagcccat cttccca 27 104 19 DNA ArtificialSequence Synthetic oligonucleotide probe 104 ctggcggtgt cctctcctt 19 10521 DNA Artificial Sequence Synthetic oligonucleotide probe 105cctcggtctc ctcatctgtg a 21 106 20 DNA Artificial Sequence Syntheticoligonucleotide probe 106 tggcccagct gacgagccct 20 107 21 DNA ArtificialSequence Synthetic oligonucleotide probe 107 ctcataggca ctcggttctg g 21108 19 DNA Artificial Sequence Synthetic oligonucleotide probe 108tggctcccag cttggaaga 19 109 30 DNA Artificial Sequence Syntheticoligonucleotide probe 109 cagctcttgg ctgtctccag tatgtaccca 30 110 21 DNAArtificial Sequence Synthetic oligonucleotide probe 110 gatgcctctgttcctgcaca t 21 111 48 DNA Artificial Sequence Synthetic oligonucleotideprobe 111 ggattctaat acgactcact atagggctgc ccgcaacccc ttcaactg 48 112 48DNA Artificial Sequence Synthetic oligonucleotide probe 112 ctatgaaattaaccctcact aaagggaccg cagctgggtg accgtgta 48 113 43 DNA ArtificialSequence Synthetic oligonucleotide probe 113 ggattctaat acgactcactatagggccgc cccgccacct cct 43 114 48 DNA Artificial Sequence Syntheticoligonucleotide probe 114 ctatgaaatt aaccctcact aaagggactc gagacaccacctgaccca 48 115 48 DNA Artificial Sequence Synthetic oligonucleotideprobe 115 ggattctaat acgactcact atagggccca aggaaggcag gagactct 48 116 48DNA Artificial Sequence Synthetic Oligonucleotide probe 116 ctatgaaattaaccctcact aaagggacta gggggtggga atgaaaag 48 117 48 DNA ArtificialSequence Synthetic oligonucleotide probe 117 ggattctaat acgactcactatagggcccc cctgagctct cccgtgta 48 118 48 DNA Artificial SequenceSynthetic oligonucleotide probe 118 ctatgaaatt aaccctcact aaagggaaggctcgccactg gtcgtaga 48 119 48 DNA Artificial Sequence Syntheticoligonucleotide probe 119 ggattctaat acgactcact atagggcaag gagccgggacccaggaga 48 120 47 DNA Artificial Sequence Synthetic oligonucleotideprobe 120 ctatgaaatt aaccctcact aaagggaggg ggcccttggt gctgagt 47

What is claimed is:
 1. Isolated nucleic acid having at least 80% nucleicacid sequence identity to a nucleotide sequence that encodes an aminoacid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6(SEQ ID NO:15), FIG. 10 (SEQ ID NO:24), FIG. 14 (SEQ ID NO:32), FIG. 16(SEQ ID NO:37), FIG. 18 (SEQ ID NO:42), FIG. 20 (SEQ ID NO:50), FIG. 22(SEQ ID NO:55), FIG. 24 (SEQ ID NO:61), FIG. 26 (SEQ ID NO:69), FIG. 28(SEQ ID NO:76), FIG. 30 (SEQ ID NO:78), FIG. 32 (SEQ ID NO:83) and FIG.34 (SEQ ID NO:91).
 2. Isolated nucleic acid having at least 80% nucleicacid sequence identity to a nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. I (SEQ ID NO:1),FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:14), FIG. 9 (SEQ ID NO:23), FIG.13 (SEQ ID NO:31), FIG. 15 (SEQ ID NO:36), FIG. 17 (SEQ ID NO:41), FIG.19 (SEQ ID NO:49), FIG. 21 (SEQ ID NO:54), FIG. 23 (SEQ ID NO:60), FIG.25 (SEQ ID NO:68), FIG. 27 (SEQ ID NO:75), FIG. 29 (SEQ ID NO:77), FIG.31 (SEQ ID NO:82) and FIG. 33 (SEQ ID NO:90).
 3. Isolated nucleic acidhaving at least 80% nucleic acid sequence identity to a nucleotidesequence selected from the group consisting of the full-length codingsequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG.3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:14), FIG. 9 (SEQ ID NO:23), FIG. 13(SEQ ID NO:31), FIG. 15 (SEQ ID NO:36), FIG. 17 (SEQ ID NO:41), FIG. 19(SEQ ID NO:49), FIG. 21 (SEQ ID NO:54), FIG. 23 (SEQ ID NO:60), FIG. 25(SEQ ID NO:68), FIG. 27 (SEQ ID NO:75), FIG. 29 (SEQ ID NO:77), FIG. 31(SEQ ID NO:82) and FIG. 33 (SEQ ID NO:90).
 4. Isolated nucleic acidhaving at least 80% nucleic acid sequence identity to the full-lengthcoding sequence of the DNA deposited under ATCC accession number 209526,209508, 209524, 209528, 209530, 209523, 209492, 209532, 209531, 209529,209527, 209570, 209618, 209621 or
 209619. 5. A vector comprising thenucleic acid of any one of claims 1 to
 4. 6. The vector of claim 5operably linked to control sequences recognized by a host celltransformed with the vector.
 7. A host cell comprising the vector ofclaim
 5. 8. The host cell of claim 7, wherein said cell is a CHO cell.9. The host cell of claim 7, wherein said cell is an E. coli.
 10. Thehost cell of claim 7, wherein said cell is a yeast cell.
 11. A processfor producing a PRO polypeptides comprising culturing the host cell ofclaim 7 under conditions suitable for expression of said PRO polypeptideand recovering said PRO polypeptide from the cell culture.
 12. Anisolated polypeptide having at least 80% amino acid sequence identity toan amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG.6 (SEQ ID NO:15), FIG. 10 (SEQ ID NO:24), FIG. 14 (SEQ ID NO:32), FIG.16 (SEQ ID NO:37), FIG. 18 (SEQ ID NO:42), FIG. 20 (SEQ ID NO:50), FIG.22 (SEQ ID NO:55), FIG. 24 (SEQ ID NO:61), FIG. 26 (SEQ ID NO:69), FIG.28 (SEQ ID NO:76), FIG. 30 (SEQ ID NO:78), FIG. 32 (SEQ ID NO:83) andFIG. 34 (SEQ ID NO:91).
 13. An isolated polypeptide scoring at least 80%positives when compared to an amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:15), FIG. 10 (SEQ IDNO:24), FIG. 14 (SEQ ID NO:32), FIG. 16 (SEQ ID NO:37), FIG. 18 (SEQ IDNO:42), FIG. 20 (SEQ ID NO:50), FIG. 22 (SEQ ID NO:55), FIG. 24 (SEQ IDNO:61), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:76), FIG. 30 (SEQ IDNO:78), FIG. 32 (SEQ ID NO:83) and FIG. 34 (SEQ ID NO:91).
 14. Anisolated polypeptide having at least 80% amino acid sequence identity toan amino acid sequence encoded by the full-length coding sequence of theDNA deposited under ATCC accession number 209526, 209508, 209524,209528, 209530, 209523, 209492, 209532, 209531, 209529, 209527, 209570,209618, 209621 or
 209619. 15. A chimeric molecule comprising apolypeptide according to any one of claims 12 to 14 fused to aheterologous amino acid sequence.
 16. The chimeric molecule of claim 15,wherein said heterologous amino acid sequence is an epitope tagsequence.
 17. The chimeric molecule of claim 15, wherein saidheterologous amino acid sequence is a Fc region of an immunoglobulin.18. An antibody which specifically binds to a polypeptide according toany one of claims 12 to
 14. 19. The antibody of claim 18, wherein saidantibody is a monoclonal antibody, a humanized antibody or asingle-chain antibody.
 20. Isolated nucleic acid having at least 80%nucleic acid sequence identity to: (a) a nucleotide sequence encodingthe polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7),FIG. 6 (SEQ ID NO:15), FIG. 10 (SEQ ID NO:24), FIG. 14 (SEQ ID NO:32),FIG. 16 (SEQ ID NO:37), FIG. 18 (SEQ ID NO:42), FIG. 20 (SEQ ID NO:50),FIG. 22 (SEQ ID NO:55), FIG. 24 (SEQ ID NO:61), FIG. 26 (SEQ ID NO:69),FIG. 28 (SEQ ID NO:76), FIG. 30 (SEQ ID NO:78), FIG. 32 (SEQ ID NO:83)or FIG. 34 (SEQ ID NO:91), lacking its associated signal peptide; (b) anucleotide sequence encoding an extracellular domain of the polypeptideshown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ IDNO:15), FIG. 10 (SEQ ID NO:24), FIG. 14 (SEQ ID NO:32), FIG. 16 (SEQ IDNO:37), FIG. 18 (SEQ ID NO:42), FIG. 20 (SEQ ID NO:50), FIG. 22 (SEQ IDNO:55), FIG. 24 (SEQ ID NO:61), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ IDNO:76), FIG. 30 (SEQ ID NO:78), FIG. 32 (SEQ ID NO:83) or FIG. 34 (SEQID NO:91), with its associated signal peptide; or (c) a nucleotidesequence encoding an extracellular domain of the polypeptide shown inFIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:15), FIG.10 (SEQ ID NO:24), FIG. 14 (SEQ ID NO:32), FIG. 16 (SEQ ID NO:37), FIG.18 (SEQ ID NO:42), FIG. 20 (SEQ ID NO:50), FIG. 22 (SEQ ID NO:55), FIG.24 (SEQ ID NO:61), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:76), FIG.30 (SEQ ID NO:78), FIG. 32 (SEQ ID NO:83) or FIG. 34 (SEQ ID NO:91),lacking its associated signal peptide.
 21. An isolated polypeptidehaving at least 80% amino acid sequence identity to: (a) the polypeptideshown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ IDNO:15), FIG. 10 (SEQ ID NO:24), FIG. 14 (SEQ ID NO:32), FIG. 16 (SEQ IDNO:37), FIG. 18 (SEQ ID NO:42), FIG. 20 (SEQ ID NO:50), FIG. 22 (SEQ IDNO:55), FIG. 24 (SEQ ID NO:61), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ IDNO:76), FIG. 30 (SEQ ID NO:78), FIG. 32 (SEQ ID NO:83) or FIG. 34 (SEQID NO:91), lacking its associated signal peptide; (b) an extracellulardomain of the polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ IDNO:7), FIG. 6 (SEQ ID NO:15), FIG. 10 (SEQ ID NO:24), FIG. 14 (SEQ IDNO:32), FIG. 16 (SEQ ID NO:37), FIG. 18 (SEQ ID NO:42), FIG. 20 (SEQ IDNO:50), FIG. 22 (SEQ ID NO:55), FIG. 24 (SEQ ID NO:61), FIG. 26 (SEQ IDNO:69), FIG. 28 (SEQ ID NO:76), FIG. 30 (SEQ ID NO:78), FIG. 32 (SEQ IDNO:83) or FIG. 34 (SEQ ID NO:91), with its associated signal peptide; or(c) an extracellular domain of the polypeptide shown in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:15), FIG. 10 (SEQ IDNO:24), FIG. 14 (SEQ ID NO:32), FIG. 16 (SEQ ID NO:37), FIG. 18 (SEQ IDNO:42), FIG. 20 (SEQ ID NO:50), FIG. 22 (SEQ ID NO:55), FIG. 24 (SEQ IDNO:61), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:76), FIG. 30 (SEQ IDNO:78), FIG. 32 (SEQ ID NO:83) or FIG. 34 (SEQ ID NO:91), lacking itsassociated signal peptide.